ATM kinase modulation for screening and therapies

ABSTRACT

The present invention relates to identification of the consensus sequence phosphorylated by ATM kinase. This, in turn, permitted identification of ATM kinase target proteins, and development of a convenient assay system for ATM kinase phosphorylation using fusion polypeptides as substrates. The assay system is adaptable to screening for ATM modulators, particularly inhibitors. In a specific embodiment, the substrate recognition sequence and mutagenized variants of this sequence were incorporated in a GST fusion protein and assayed for phosphorylation by ATM kinase. This assay system is useful in screening for ATM inhibitors. ATM function assays were validated using an ATM-kinase dead dominant-negative mutant.

[0001] This is a division of application Ser. No. 09/400,653, filed Sep.21, 1999, which is a continuation-in-part of and claims the priority ofU.S. application Ser. No. 09/248,061 filed Feb. 10, 1999. Each of theseprior applications is hereby incorporated herein by reference, in itsentirety.

[0002] The research leading to the present invention was supported, inpart, by National Institute of Health grants CA71387 and ES05777.Accordingly, the Government has certain rights in the invention.

FIELD OF THE INVENTION

[0003] The present invention relates to identification of the consensussequence phosphorylated by ATM kinase. This, in turn, permittedidentification of ATM kinase target proteins, and development of aconvenient assay system for ATM kinase phosphorylation using fusionpolypeptides as substrates. The assay system is adaptable to screeningfor ATM modulators, particularly inhibitors.

BACKGROUND OF THE INVENTION

[0004] Ataxia telangiectasia (AT) is a rare autosomal recessivemulti-system disorder characterized by clinical manifestations thatinclude progressive cerebellar ataxia, neuronal degeneration,hypersensitivity to ionizing irradiation (IR), premature aging,hypogonadism, growth retardation, immune deficiency, and an increasedrisk for cancer (Lavin and Shiloh, Annu. Rev. Immunol., 15:177, 1997).Cancer predisposition in AT is striking: 38% of patients developmalignancies, mainly lymphoreticular neoplasms and leukemias. But ATpatients manifest acute radiosensitivity and must be treated withreduced radiation doses, and not with radiomimetic chemotherapy. AT hasa worldwide frequency of 1:100,000 live births and an estimated carrierfrequency of 1% in the American population. Notable concentrations of ATpatients outside the United States are in Turkey, Italy, and Israel.

[0005] Cerebellar ataxia that gradually develops into general motordysfunction is the first clinical hallmark and results from progressiveloss of Purkinje cells in the cerebellum. Oculocutaneous telangiectasia(dilation of blood vessels) develops in the bulbar conjunctiva andfacial skin, and is later accompanied by graying of the hair andatrophic changes in the skin. Somatic growth is retarded in mostpatients, and ovarian dysgenesis is typical for female patients. Amongoccasional endocrine abnormalities, insulin-resistant diabetes ispredominant, and serum levels of alpha-fetoprotein and carcinoembryonicantigen are elevated. The thymus is either absent or vestigial, andother immunological defects include reduced levels of serum IgA, IgE orIgG2, peripheral lymphopenia, and reduced responses to viral antigensand allogeneic cells. These immunological defects cause many patients tosuffer from recurrent sinopulmonary infections. The most common cause ofdeath in AT, typically during the second or third decade of life, isfrom these sinopulmonary infections with or without malignancy.

[0006] The gene mutated in AT, ATM (Ataxia Telangiectasia-Mutated),encodes a 370-kD protein that is a member of a family of proteinsrelated to phosphatidylinositol 3-kinase (PI-3-K) that have either lipidor protein kinase activity. A subset of this family with the greatesthomology to ATM functions in DNA repair, DNA recombination, and cellcycle control (Savitsky et al., Science, 268:1749, 1995; Keith andScreiber, ibid., 270:50, 1995). Cell lines derived from AT patientsexhibit hypersensitivity to ionizing radiation (IR) and defects inseveral IR-inducible cell cycle checkpoints, including a diminishedirradiation-induced arrest in the G1 phase of the cell cycle mediated bythe p53 tumor suppressor gene product (Kastan et al., Cell, 71:587,1992; Morgan and Kastan, Adv. Cancer Res., 71:1, 1997). In response toDNA damage, cells with wild type ATM accumulate p53 protein and show asubsequent increase in p53 activity, whereas cells with defective ATMshow a smaller increase in the amount of p53 protein in response to IR(Kastan et al., supra; Canman et al., Cancer Res., 54:5054, 1994; Khannaand Lavin, Oncogene, 8:3307, 1993). Therefore, ATM appears to actupstream of p53 in a signal transduction pathway initiated by IR.

[0007] p95/nibrin has recently been shown to be the gene mutated in theNijmegen breakage syndrome (NBS), which is an autosomal recessivedisease with a phenotype (radiation sensitivity, predisposition tomalignancies, and chromosomal instability) virtually identical to thatof AT (Shiloh, 1997; Carney et al., 1998; Varon et al., 1998;Featherstone and Jackson, 1998). The main distinction between the AT andNBS syndromes is that AT patients exhibit progressive ataxia while NBSpatients have microcephaly as their neurologic abnormality. p95/nibrinis part of a double-strand break DNA repair protein complex containingRad50 and MRE11 (Carney et al., 1998; Dong et al., 1999; Paul andGellert, 1999). The overlapping phenotypes of AT and NBS suggest thatATM and p95/nibrin may play a role in the same cellular pathways, likelyincluding roles in responses to DNA damage. Identifying p95/nibrin as anin vitro ATM kinase target led us to investigate potential in vivointeractions between p95/nibrin and ATM. We found that ATM protein canbind to p95/nibrin and that ATM activity is required for phosphorylationof p95 on Ser343 after IR. Thus, characterization of the ATM kinase andelucidation of in vitro targets led to identification of one validphysiologically significant target and will likely lead to furtherinsights into ATM function and AT biology.

[0008] IR induces rapid, de novo phosphorylation of endogenous p53 attwo serine residues within the first 24 amino acids of the protein, oneof which was identified as Ser¹⁵ (Shieh et el., Cell, 91:325, 1997;Siliciano et al., Genes Dev., 11:3471, 1997). Phosphorylation of p53 atSer¹⁵ in response to DNA damage correlates with both the accumulation oftotal p53 protein as well as with the ability of p53 to transactivatedownstream target genes in wild type cells (Siliciano et al., supra).Furthermore, phosphorylation of p53 on Ser¹⁵ in response to IR isdiminished in cell lines derived from AT patients, suggesting that ATMparticipates in this response (Siliciano et al., supra).

[0009] The P13-K-related protein, DNA-activated protein kinase (DNA-PK),phosphorylates p53 in vitro at two different SQ motifs, Ser¹⁵ and Ser³⁷(Lees-Miller et al., Mol. Cell Biol., 12:5041, 1992). However, cellswith diminished DNA-PK activity still normally accumulate p53 proteinand undergo G1 arrest in response to IR (Rathmell et al., Cancer Res.,57:68, 1997; Guialos et al, Genes Dev., 10:2038, 1996; Nacht et al.,ibid., p. 2055).

[0010] The concept of inhibiting ATM for the treatment of neoplasms,particularly cancers associated with decreased p53 function, has beensuggested (Morgan et al., Mol. Cell Biol. 17:2020, 1997; Hartwell andKastan, Science, 266:1821, 1994; Kastan, New Eng. J. Med. 333:662, 1995;see also WO 98/56391, Westphal and Leder). In particular, Westphal andLeder provide genetically manipulated knock-out mice as a model fortesting ATM inhibitors. This published application suggests using aninhibitory antibody to ATM, a dominant negative fragment of ATM (seealso Morgan et al., supra), or an ATM antisense strategy to inhibit ATM.However, while these publications propose inhibiting ATM to enhanceradiosensitivity of neoplastic cells, and screening for compounds thatinhibit ATM activity, they provide no specific screening test,particularly one suitable for high through-put screening. There is nohint or suggestion in these publications of strategies for targeting thekinase activity of ATM, the nature of an ATM kinase substraterecognition sequence, or of sequences recognized specifically by ATM,but not other kinases. There is also no information about the functionof other ATM target proteins besides p53.

[0011] Accordingly, there is a need in the art to understand ATM kinasespecificity. There is a further need to identify ATM target proteinsother than p53.

[0012] These and other needs in the art are addressed by the presentinvention.

SUMMARY OF THE INVENTION

[0013] The present invention relates to the identification of an ATMkinase substrate recognition consensus sequence motif, and to theidentification of new ATM target proteins, which in turn has led to thediscovery of unexpected and novel ATM-regulated cellular pathways.

[0014] Thus, in one embodiment, the invention advantageously provides amethod for identifying an ATM kinase substrate recognition sequence in aprotein. This method comprises contacting an ATM kinase with a fusionpolypeptide and detecting whether binding has occurred between the ATMkinase and the fusion polypeptide. The fusion polypeptide contains astructural portion and a candidate ATM-kinase substrate recognitionsequence portion. Moreover, given application of sequence comparisontechniques, the invention provides a method for identifying a putativeATM target protein, by analyzing the sequence of the protein todetermine whether it contains an ATM substrate recognition consensussequence motif.

[0015] In a further embodiment, a method for identifying anATM-regulated pathway is provided. This method comprises identifying asubstrate of an ATM kinase, e.g., as described above; modulatingATM-mediated phosphorylation of the target protein comprising an ATMrecognition sequence; and determining whether modulation of ATM-mediatedphosphorylation of the target protein affects a cellular pathway, whichwould indicate that the pathway is an ATM-regulated pathway. As acorollary, the invention provides a method for modulating anATM-regulated pathway, which comprises modulating ATM-mediatedphosphorylation of a target protein comprising an ATM-kinase recognitionsequence in a cell.

[0016] The methods of the invention can involve a kinase-dead ATM mutantpolypeptide. Thus, in a further embodiment, the invention provides anucleic acid encoding such a polypeptide, as well as the polypeptideitself. The invention provides a recombinant vector which codes forexpression of a defective ATM polypeptide, e.g., a kinase dead mutant,and a cell line containing such a vector.

[0017] In another embodiment, particularly in connection with methodsfor identifying an ATM kinase substrate recognition sequence and forscreening, the invention provides a fusion polypeptide, wherein thefusion polypeptide contains a structural portion and an ATM-kinaserecognition sequence portion.

[0018] In still another embodiment, the invention provides a method forscreening for a compound that modulates ATM-mediated phosphorylation.This method comprises detecting whether there is a change in the levelof ATM-mediated phosphorylation of a polypeptide comprising an ATMsubstrate recognition sequence in the presence of a candidate compound,wherein an increase in the level of phosphorylation indicates that thecompound agonizes ATM-mediated phosphorylation, and a decrease in thelevel of phosphorylation indicates that the compound antagonizesATM-mediated phosphorylation.

[0019] The invention further provides for screening for a compound thatinduces an ATM-regulated pathway in a cell, comprising contacting thecell with a candidate compound, and detecting whether the ATM-mediatedpathway is induced in the cell, wherein the cell is defective forexpression of ATM. In one embodiment, the screening can be forATM-regulated cellular pathway, with the proviso that the pathway doesnot involve p53 or cell cycle control, or both.

[0020] The invention also provides methods for modulating ATM kinaseactivity in cells in vitro and in vivo. Such modulation includesinhibition of ATM kinase. In vivo modulation provides for evaluation ofATM function, e.g., in animal models. Alternatively, in vivo modulationof ATM function has therapeutic effects. In particular, ATM inhibitioncan enhance radiosensitivity and chemotherapeutic sensitivity in tumors,inhibit cell proliferation and induce revascularization in restenosis,and promote insulin signaling and increased metabolism in obesity.

[0021] In yet another embodiment, the invention provides a compositioncomprising ATM and a polypeptide, in which the polypeptide comprises anATM kinase substrate recognition sequence, e.g., for co-crystallizationor other methods of structure-function analysis. The results of suchstructural studies permit rational drug design and development.

[0022] These and other aspects of the present invention are furtherelaborated in the Detailed Description of the Invention and Examples,infra.

DESCRIPTION OF THE DRAWINGS

[0023]FIGS. 1A, 1B, and 1C. Phosphorylation of GST-p53 mutant peptides(amino acids 9-21) by ATM, ATR, and DNA-PK in vitro. In vitro kinaseassays with immunoprecipitated, transfected flag-tagged ATM (A) or ATR(B) or purified DNA-PK (C) were performed with GST-p53 peptidescontaining the amino acid substitutions indicated. The amount of32P-labeled GST-peptides was quantitated with a Phosphorlmager and wasnormalized for each of the kinases to the level of phosphorylation ofwild-type GST-p53.

[0024]FIGS. 2A, 2B, and 2C. Phosphorylation of candidate targets of byATM, ATR and DNA-PK in vitro. GST-peptides containing the putativetarget sequences were used as substrates for the kinase assay. The aminoacid sequences of the GST-peptides are as indicated in Table 4.

[0025]FIG. 3. p95 binds to ATM and IR induces phosphorylation of p95 innormal cells, resulting in mobility shift of p95 following IR in normal,but not AT, cells. Normal cells (TK6, K562) or AT cells (AT3LA) weretreated with either 0 (−) or 10 (+) Gy irradiation and p95 wasimmunoprecipitated, either not treated (−) or treated (+) with lambdaphosphatase, and analyzed by SDS-PAGE and western blotting for p95. Thetop arrowhead indicates the slower mobility band.

[0026]FIGS. 4A and 4B. In vivo phosphorylation of p95 on ser343 by ATMupon ionizing irradiation. (A) Phosphorylation of p95 on Ser343 by IR inan ATM-dependent manner in vivo. Myc-p95 was co-transfected into 293Tcells with either empty vector or wild-type (wt) or kinase inactive (kd)Flag-ATM and cells were treated with either 0 (−) or 5 (+) Gy IR asindicated. The expression of Flag-ATM in each cell lysates was detectedwith anti-Flag antibody (top panel). Immunoprecipitated Myc-p95 wasblotted with anti-a-p95-phosphoserine 343 antibody (middle panel) oranti-Myc antibody (lower panel). (B) Phosphorylation of endogenous p95on Ser343 in normal cells, but not AT cells, after IR. Normal (WT,GM0536) or AT (GM1526) lymphoblasts were treated with either 0 (−) or 5(+) Gy IR. Endogenous p95 was immunoprecipitated and blotted withanti-a-p95-phosphoserine 343 antibody (top panel) or anti-p95 antibody(lower panel).

DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention is based, in part, on the discovery ofsubstrate recognition sequences of ATM kinase target proteins. Theinitial discovery was made using p53 as the target protein. Utilizingsite-directed mutagenesis, the kinase recognition sequence in p53 wasdissected, and the critical residues identified. Additional putative ATMkinase recognition sites were identified in other proteins, and thesesequences were tested in a fusion polypeptide assay for the ability tobe phosphorylated by ATM kinase. Using a reiterative process, aconsensus sequence motif, termed herein an ATM kinase substraterecognition consensus sequence motif, has been determined.

[0028] In addition, kinase specificity has been evaluated, anddistinctions between different kinases (ATM, DNA-PK, and ATR) have beenfound.

[0029] Coincident with characterization of the recognition consensussequence motif, an assay for kinase activity was developed. In thepresent invention, a full-length ATM protein is expressed and used inphosphorylation assays, in which the presence of manganese has beenfound to be essential. Furthermore, a dominant-negative ATM kinasemutant was developed as a negative control for the assay. The assay usedto characterize the ATM kinase substrate recognition consensus sequencemotif further involves creating chimeric polypeptides comprising aputative ATM kinase substrate recognition sequence, which can be testedas phosphorylation substrates for the full-length ATM. The assay can beadapted for a number of purposes.

[0030] First, putative ATM kinase recognition sites from possible targetproteins can be inserted in the ATM kinase substrate polypeptide andtested for phosphorylation by ATM. Thus, probable physiological targetsof ATM phosphorylation can be identified. Where the cellular processesthat these targets affect are known, this assay can establish alikelihood that the cellular process is regulated, at least in part, byATM.

[0031] Second, the ATM kinase substrate polypeptides can be used toscreen for ATM kinase agonists or antagonists. Since the chimericpolypeptides have a structural portion, which may provide for specificbinding, they can be readily evaluated for phosphorylation. Thus,screening assays for compounds that agonize or antagonize ATM activitycan be performed by measuring the level of phosphorylation of the ATMsubstrate polypeptide, e.g., either with in vitro cell-free or cellularassay methods. Recombinant expression systems can be used to express ATMand ATM substrate polypeptides for cell-free, in vitro assays.Alternatively, cells that express ATM (either endogenously orrecombinantly) are engineered to express the ATM substrate polypeptide,so that any physiological effects of the ATM substrate polypeptide oncellular processes can be evaluated.

[0032] Third, the ATM substrate polypeptide can be used as a competitiveinhibitor of ATM phosphorylation in cellular assays. In one embodiment,the ATM kinase recognition site sequence of the polypeptide isspecifically recognized by ATM, but not by DNA-PK, ATR, or otherkinases. In another embodiment, the presence of a kinase recognitionsequence in the ATM substrate polypeptide only inhibits phosphorylationof the specific target protein from which the sequence was derived, thusspecifically affecting one ATM-regulated process. In still anotherembodiment, the ATM substrate polypeptide has a substrate sequence thatis generally recognized by ATM and another kinase or kinases.

[0033] In another embodiment, the invention provides ATM functionalassays. A stably expressed, dominant-negative kinase dead ATM mutant ofthe invention can be used to validate these assays, and further permitsevaluation of the cellular processes regulated by ATM. Thedominant-negative mutant, a competitive inhibitor ATM substratepolypeptide, or an ATM antagonist (or agonist) compound discovered usingthe screening assays described herein, can be used in assays for ATMfunction, including in vitro cell-based assays for cell cycle processes,radiation sensitivity, and chemotherapeutic sensitivity, and animalmodels of ATM function. These cell-based and animal models have directcorrelates with therapeutic methods based on modulation, e.g.,inhibition, of ATM activity. Inhibition of ATM activity clearly isimportant in treating tumors, by rendering the tumors more sensitive toradiation or chemotherapeutic agents. Although the prior art hassuggested a role for inhibition of ATM in these processes, thediscoveries of the present invention permit a detailed understanding ofthe mechanism by which ATM affects these processes, which in turnpermits a more effective, rational approach to targeting therapy fortumors without inhibiting physiologically beneficial ATM activity.Furthermore, these results have unexpected implications for additionaltherapeutic modalities, including enhancing anti-restenosis strategiesbased on inhibition of cellular proliferation. The present inventionfurther provides a strategy for targeting ATM-mediated insulin signaltransduction by PHASI in adipocytes, for treating obesity. It should benoted that the therapeutic aspects of the invention can be directlyevaluated in the animal model assays described herein, and that theanimal models substantiate therapeutic potential of ATM modulation.

[0034] Thus, the present invention permits more precise analysis ofknown and discovered ATM-regulated cellular process. The term “cellularprocess” is used herein to refer to cellular processes, such as, but byno means limited to, double stranded DNA break repair, telomeresynthesis or repair, the aging process, tumor suppression, insulin andinsulin-like growth factor (IGF)-I signaling, cell cycle control, cellsurvival after HTLV infection, and autophosphorylation. The inventionhas advantageously permitted identification of novel cellular processesthat are regulated by ATM-mediated phosphorylation of various targetingproteins, such as DNA repair, aging, neurodegeneration, HTLV infection,and significantly, obesity. Furthermore, the discoveries of theinvention permit more precise and complete biochemical analysis ofprocesses, such as tumor suppression in conjunction with ionizingradiation and cell cycling. The term “ATM-regulated process” is usedherein to refer to a cellular process that depends upon or is affectedby ATM-mediated phosphorylation of a target protein.

[0035] Accordingly, more extensive descriptions of the various aspectsof the invention are provided in the following sections of theapplication: ATM kinase (including kinase dead mutants); the ATM kinasesubstrate recognition consensus sequence (including disclosure of ATMtarget proteins that have this motif); ATM substrate polypeptides (thefusion polypeptides for testing whether a motif sequence isphosphorylated by ATM and for screening assays); crystal structure ofATM-ATM kinase substrate recognition sequence complexes (for rationaldrug design); recombinant expression systems (for screening, functionassays, and therapeutics); screening assays (for identification ofmodulators of ATM function); assays for ATM function (usingdominant-negative ATM and ATM modulator compounds); and modulation ofATM activity for therapy. The headings (bold), subheadings (bold,italics), and sections of the application are provided to facilitateunderstanding of the invention, and are not intended to be limiting.

General Definitions

[0036] In a specific embodiment, the term “about” or “approximately”means within 20%, preferably within 10%, and more preferably within 5%of a given value or range.

[0037] As used herein, the term “isolated” means that the referencedmaterial is free of components found in the natural environment in whichthe material is normally found. In particular, isolated biologicalmaterial is free of cellular components. In the case of nucleic acidmolecules, an isolated nucleic acid includes a PCR product, an isolatedmRNA, a cDNA, or a restriction fragment. In another embodiment, anisolated nucleic acid is preferably excised from the chromosome in whichit may be found, and more preferably is no longer joined tonon-regulatory, non-coding regions, or to other genes, located upstreamor downstream of the gene contained by the isolated nucleic acidmolecule when found in the chromosome. In yet another embodiment, theisolated nucleic acid lacks one or more introns. Isolated nucleic acidmolecules can be inserted into plasmids, cosmids, artificialchromosomes, and the like. Thus, in a specific embodiment, a recombinantnucleic acid is an isolated nucleic acid. An isolated protein may beassociated with other proteins or nucleic acids, or both, with which itassociates in the cell, or with cellular membranes if it is amembrane-associated protein. An isolated organelle, cell, or tissue isremoved from the anatomical site in which it is found in an organism. Anisolated material may be, but need not be, purified.

[0038] The term “purified” as used herein refers to material that hasbeen isolated under conditions that reduce or eliminate unrelatedmaterials, i.e., contaminants. For example, a purified protein ispreferably substantially free of other proteins or nucleic acids withwhich it is associated in a cell; a purified nucleic acid molecule ispreferably substantially free of proteins or other unrelated nucleicacid molecules with which it can be found within a cell. A purifiedtumor cell is preferably substantially free of other normal cells. Asused herein, the term “substantially free” is used operationally, in thecontext of analytical testing of the material. Preferably, purifiedmaterial substantially free of contaminants is at least 50% pure; morepreferably, at least 90% pure, and more preferably still at least 99%pure. Purity can be evaluated by chromatography, gel electrophoresis,immunoassay, composition analysis, biological assay, and other methodsknown in the art.

[0039] The use of italics (e.g., ATM) indicates a nucleic acid molecule(cDNA, mRNA, gene, etc.); normal text (e.g., ATM) indicates thepolypeptide or protein.

Molecular Biology Definitions

[0040] In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, New York (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds.(1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins,eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)];Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, APractical Guide To Molecular Cloning (1984); F. M. Ausubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994).

[0041] Therefore, if appearing herein, the following terms shall havethe definitions set out below.

[0042] The term “host cell” means any cell of any organism that isselected, modified, transformed, grown, or used or manipulated in anyway, for the production of a substance by the cell, for example theexpression by the cell of a gene, a DNA or RNA sequence, a protein or anenzyme. Host cells can further be used for screening or functionalassays, as described infra. A host cell has been “transfected” byexogenous or heterologous DNA when such DNA has been introduced insidethe cell. A cell has been “transformed” by exongenous or heterologousDNA when the transfected DNA is expressed and effects a function orphenotype on the cell in which it is expressed. The term “expressionsystem” means a host cell transformed by a compatible expression vectorand cultured under suitable conditions e.g. for the expression of aprotein coded for by foreign DNA carried by the vector and introduced tothe host cell.

[0043] Proteins and polypeptides can be made in the host cell byexpression of recombinant DNA. As used herein, the term “polypeptide”refers to an amino acid-based polymer, which can be encoded by a nucleicacid or prepared synthetically. Polypeptides can be proteins, proteinfragments, chimeric proteins, etc. Generally, the term “protein” refersto a polypeptide expressed endogenously in a cell, e.g., the naturallyoccuring form (or forms) of the amino acid-based polymer. Thus, thephysiological substrate of ATM kinase is a “target protein”, while achimeric construct comprising an ATM kinase substrate recognitionsequence is a “fusion polypeptide.”

[0044] A “coding sequence” or a sequence “encoding” an expressionproduct, such as a RNA, polypeptide, protein, or enzyme, is a nucleotidesequence that, when expressed, results in the production of that RNA,polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodesan amino acid sequence for that polypeptide, protein or enzyme. A codingsequence for a protein may include a start codon (usually ATG) and astop codon.

[0045] The coding sequences herein may be flanked by natural regulatory(expression control) sequences, or may be associated with heterologoussequences, including promoters, internal ribosome entry sites (IRES) andother ribosome binding site sequences, enhancers, response elements,suppressors, signal sequences, polyadenylation sequences, introns, 5′-and 3′-non-coding regions, and the like. The nucleic acids may also bemodified by many means known in the art. Non-limiting examples of suchmodifications include methylation, “caps”, substitution of one or moreof the naturally occurring nucleotides with an analog, andinternucleotide modifications.

[0046] The term “gene”, also called a “structural gene” means a DNAsequence that codes for or corresponds to a particular sequence ofribonucleic acids or amino acids which comprise all or part of one ormore proteins, and may or may not include regulatory DNA sequences, suchas promoter sequences, which determine for example the conditions underwhich the gene is expressed.

[0047] A “promoter sequence” is a DNA regulatory region capable ofbinding RNA polymerase in a cell and initiating transcription of adownstream (3′ direction) coding sequence. For purposes of defining thepresent invention, the promoter sequence is bounded at its 3′ terminusby the transcription initiation site and extends upstream (5′ direction)to include the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background.

[0048] A coding sequence is “under the control” or “operativelyassociated with” of transcriptional and translational control sequencesin a cell when RNA polymerase transcribes the coding sequence into mRNA,which then may be trans-RNA spliced (if it contains introns) andtranslated into the protein encoded by the coding sequence.

[0049] The terms “express” and “expression” mean allowing or causing theinformation in a gene or DNA sequence to become manifest, for exampleproducing a protein by activating the cellular functions involved intranscription and translation of a corresponding gene or DNA sequence. ADNA sequence is expressed in or by a cell to form an “expressionproduct” such as a protein. The expression product itself, e.g. theresulting protein, may also be said to be “expressed” by the cell.

[0050] The term “transfection” means the introduction of a foreignnucleic acid into a cell. The term “transformation” means theintroduction of a “foreign” (i.e. extrinsic or extracellular) gene, DNAor RNA sequence to a host cell, so that the host cell will express theintroduced gene or sequence to produce a desired substance, typically aprotein or enzyme coded by the introduced gene or sequence. Theintroduced gene or sequence may also be called a “cloned”, “foreign”, or“heterologous” gene or sequence, and may include regulatory or controlsequences used by a cell's genetic machinery. The gene or sequence mayinclude nonfunctional sequences or sequences with no known function. Ahost cell that receives and expresses introduced DNA or RNA has been“transformed” and is a “transformant” or a “clone.” The DNA or RNAintroduced to a host cell can come from any source, including cells ofthe same genus or species as the host cell, or cells of a differentgenus or species.

[0051] The terms “vector”, “cloning vector” and “expression vector” meanthe vehicle by which a DNA or RNA sequence (e.g., a foreign gene) can beintroduced into a host cell, so as to transform the host and promoteexpression (e.g., transcription and translation) of the introducedsequence. Vectors include plasmids, phages, viruses, etc. A “cassette”refers to a DNA coding sequence or segment of DNA that codes for anexpression product that can be inserted into a vector at definedrestriction sites. The cassette restriction sites are designed to ensureinsertion of the cassette in the proper reading frame. Generally,foreign DNA is inserted at one or more restriction sites of the vectorDNA, and then is carried by the vector into a host cell along with thetransmissible vector DNA. A segment or sequence of DNA having insertedor added DNA, such as an expression vector, can also be called a “DNAconstruct.” Recombinant cloning vectors will often include one or morereplication systems for cloning or expression, one or more markers forselection in the host, e.g. antibiotic resistance, and one or moreexpression cassettes.

[0052] The term “heterologous” refers to a combination of elements notnaturally occurring. For example, heterologous DNA refers to DNA notnaturally located in the cell, or in a chromosomal site of the cell.Preferably, the heterologous DNA includes a gene foreign to the cell. Aheterologous expression regulatory element is a such an elementoperatively associated with a different gene than the one it isoperatively associated with in nature. In the context of the presentinvention, an gene is heterologous to the recombinant vector DNA inwhich it is inserted for cloning or expression, and it is heterologousto a host cell containing such a vector, in which it is expressed, e.g.,a CHO cell.

[0053] The terms “mutant” and “mutation” mean any detectable change ingenetic material, e.g. DNA, or any process, mechanism, or result of sucha change. This includes gene mutations, in which the structure (e.g.,DNA sequence) of a gene is altered, any gene or DNA arising from anymutation process, and any expression product (e.g., protein) expressedby a modified gene or DNA sequence. The term “variant” may also be usedto indicate a modified or altered gene, DNA sequence, enzyme, cell,etc., i.e., any kind of mutant.

[0054] “Sequence-conservative variants” of a polynucleotide sequence arethose in which a change of one or more nucleotides in a given codonposition results in no alteration in the amino acid encoded at thatposition.

[0055] “Function-conservative variants” are those in which a given aminoacid residue in a protein or enzyme has been changed without alteringthe overall conformation and function of the polypeptide, including, butnot limited to, replacement of an amino acid with one having similarproperties (such as, for example, polarity, hydrogen bonding potential,acidic, basic, hydrophobic, aromatic, and the like). Amino acids withsimilar properties are well known in the art. For example, arginine,histidine and lysine are hydrophilic-basic amino acids and may beinterchangeable. Similarly, isoleucine, a hydrophobic amino acid, may bereplaced with leucine, methionine or valine. Such changes are expectedto have little or no effect on the apparent molecular weight orisoelectric point of the protein or polypeptide. Amino acids other thanthose indicated as conserved may differ in a protein or enzyme so thatthe percent protein or amino acid sequence similarity between any twoproteins of similar function may vary and may be, for example, from 70%to 99% as determined according to an alignment scheme such as by theCluster Method, wherein similarity is based on the MEGALIGN algorithm. A“function-conservative variant” also includes a polypeptide or enzymewhich has at least 60% amino acid identity as determined by BLAST orFASTA algorithms, preferably at least 75%, most preferably at least 85%,and even more preferably at least 90%, and which has the same orsubstantially similar properties or functions as the native or parentprotein or enzyme to which it is compared.

[0056] As used herein, the term “homologous” in all its grammaticalforms and spelling variations refers to the relationship betweenproteins that possess a “common evolutionary origin,” including proteinsfrom superfamilies (e.g., the immunoglobulin superfamily) and homologousproteins from different species (e.g., myosin light chain, etc.) (Reecket al., Cell 50:667, 1987). Such proteins (and their encoding genes)have sequence homology, as reflected by their sequence similarity,whether in terms of percent similarity or the presence of specificresidues or motifs.

[0057] Accordingly, the term “sequence similarity” in all itsgrammatical forms refers to the degree of identity or correspondencebetween nucleic acid or amino acid sequences of proteins that may or maynot share a common evolutionary origin (see Reeck et al., supra).However, in common usage and in the instant application, the term“homologous,” when modified with an adverb such as “highly,” may referto sequence similarity and may or may not relate to a commonevolutionary origin.

[0058] In a specific embodiment, two DNA sequences are “substantiallyhomologous” or “substantially similar” when at least about 80%, and mostpreferably at least about 90 or 95% of the nucleotides match over thedefined length of the DNA sequences, as determined by sequencecomparison algorithms, such as BLAST, FASTA, DNA Strider, etc. Sequencesthat are substantially homologous can be identified by comparing thesequences using standard software available in sequence data banks, orin a Southern hybridization experiment under, for example, stringentconditions as defined for that particular system.

[0059] Similarly, in a particular embodiment, two amino acid sequencesare “substantially homologous” or “substantially similar” when greaterthan 80% of the amino acids are identical, or greater than about 90% aresimilar (functionally identical). Preferably, the similar or homologoussequences are identified by alignment using, for example, the GCG(Genetics Computer Group, Program Manual for the GCG Package, Version 7,Madison, Wisconsin) pileup program, or any of the programs describedabove (BLAST, FASTA, etc.).

[0060] A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength (see Sambrook et al., supra). For hybrids of greater than100 nucleotides in length, equations for calculating Tm have beenderived (see Sambrook et al., supra, 9.50-9.51). For hybridization withshorter nucleic acids, i.e., oligonucleotides, the position ofmismatches becomes more important, and the length of the oligonucleotidedetermines its specificity (see Sambrook et al., supra, 11.7-11.8). Aminimum length for a hybridizable nucleic acid is at least about 10nucleotides; preferably at least about 15 nucleotides; and morepreferably the length is at least about 20 nucleotides.

[0061] The present invention provides antisense nucleic acids (includingribozymes), which may be used to inhibit expression of ATM kinase, oralternatively an ATM kinase target protein, e.g., to disrupt a cellularprocess. An “antisense nucleic acid” is a single stranded nucleic acidmolecule which, on hybridizing under cytoplasmic conditions withcomplementary bases in an RNA or DNA molecule, inhibits the latter'srole. If the RNA is a messenger RNA transcript, the antisense nucleicacid is a countertranscript or mRNA-interfering complementary nucleicacid.

[0062] As presently used, “antisense” broadly includes RNA-RNAinteractions, RNA-DNA interactions, ribozymes and RNase-H mediatedarrest. Antisense nucleic acid molecules can be encoded by a recombinantgene for expression in a cell (e.g., U.S. Pat. No. 5,814,500; U.S. Pat.No. 5,811,234), or alternatively they can be prepared synthetically(e.g., U.S. Pat. No. 5,780,607).

[0063] As used herein, the term “oligonucleotide” refers to a nucleicacid, generally of at least 10, preferably at least 15, and morepreferably at least 20 nucleotides, preferably no more than 100nucleotides, that is hybridizable to a genomic DNA molecule, a cDNAmolecule, or an mRNA molecule encoding a gene, mRNA, cDNA, or othernucleic acid of interest. Oligonucleotides can be labeled, e.g., with³²P-nucleotides or nucleotides to which a label, such as biotin, hasbeen covalently conjugated. In one embodiment, a labeled oligonucleotidecan be used as a probe to detect the presence of a nucleic acid. Inanother embodiment, oligonucleotides (one or both of which may belabeled) can be used as PCR primers, e.g., for cloning full length or afragment of a protein or polypeptide. In a further embodiment, anoligonucleotide of the invention can form a triple helix with a nucleicacid (genomic DNA or mRNA) encoding a protein or polypeptide. Generally,oligonucleotides are prepared synthetically, preferably on a nucleicacid synthesizer. Accordingly, oligonucleotides can be prepared withnon-naturally occurring phosphoester analog bonds, such as thioesterbonds, etc. Furthermore, the oligonucleotides herein may also bemodified with a label capable of providing a detectable signal, eitherdirectly or indirectly. Exemplary labels include radioisotopes,fluorescent molecules, biotin, and the like.

[0064] Specific non-limiting examples of synthetic oligonucleotidesenvisioned for this invention include oligonucleotides that containphosphorothioates, phosphotriesters, methyl phosphonates, short chainalkyl, or cycloalkl intersugar linkages or short chain heteroatomic orheterocyclic intersugar linkages. Most preferred are those withCH₂—NH—O—CH₂, CH₂—N(CH₃)—O—CH₂, CH₂—O—N(CH₃)—CH₂, CH₂—N(CH₃)—N(CH₃)—CH₂and O—N(CH₃)—CH₂—CH₂ backbones (where phosphodiester is O—PO₂—O—CH₂).U.S. Pat. No. 5,677,437 describes heteroaromatic olignucleosidelinkages. Nitrogen linkers or groups containing nitrogen can also beused to prepare oligonucleotide mimics (U.S. Pat. No. 5,792,844 and No.5,783,682). U.S. Pat. No. 5,637,684 describes phosphoramidate andphosphorothioamidate oligomeric compounds. Also envisioned areoligonucleotides having morpholino backbone structures (U.S. Pat. No.5,034,506). In other embodiments, such as the peptide-nucleic acid (PNA)backbone, the phosphodiester backbone of the oligonucleotide may bereplaced with a polyamide backbone, the bases being bound directly orindirectly to the aza nitrogen atoms of the polyamide backbone (Nielsenet al., Science 254:1497, 1991). Other synthetic oligonucleotides maycontain substituted sugar moieties comprising one of the following atthe 2′ position: OH, SH, SCH₃, F, OCN, O(CH₂)_(n)NH₂ or O(CH₂)_(n)CH₃where n is from 1 to about 10; C₁ to C₁₀ lower alkyl, substituted loweralkyl, alkaryl or aralkyl; Cl; Br; CN; CF₃; OCF₃; O-; S-, or N-alkyl;O-, S-, or N-alkenyl; SOCH₃; SO₂CH₃; ONO₂;NO₂; N₃; NH₂;heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino;substitued silyl; a fluorescein moiety; an RNA cleaving group; areporter group; an intercalator; a group for improving thepharmacokinetic properties of an oligonucleotide; or a group forimproving the pharmacodynamic properties of an oligonucleotide, andother substituents having similar properties. Oligonucleotides may alsohave sugar mimetics such as cyclobutyls or other carbocyclics in placeof the pentofuranosyl group. Nucleotide units having nucleosides otherthan adenosine, cytidine, guanosine, thymidine and uridine, such asinosine, may be used in an oligonucleotide molecule.

ATM Kinase

[0065] The methods and compositions of the present invention relate, inparticular, to the activity of an ATM kinase. As used herein, the term“ATM kinase” (or “ATM”) refers to a polypeptide that phosphorylatestarget proteins that have an ATM kinase substrate recognition consensussequence motif, as described herein, or the nucleic acid (cDNA orgenomic DNA) encoding such an ATM kinase. Such ATM kinases include humanATM kinase described in U.S. Pat. No. 5,756,288, U.S. Pat. No.5,728,807, and U.S. Pat. No. 5,777,093, including both wild-type andnaturally occurring mutant ATM kinases. Naturally occurring mutant ATMkinases are either truncated or are unstable proteins. The inventionfurther advantageously provides an engineered, non-naturally occurring,kinase-dead ATM, which behaves as a dominant-negative mutant and ispreferred for use in ATM function assays as described herein. In aspecific embodiment, the dominant-negative kinase-dead mutant has anamino acid sequence assigned accession number 1585222, and is encoded bya cDNA having a nucleotide sequence assigned accession number U33841, inthe NCBI database. The term also encompasses non-human ATM kinases,which can be used in the various assays and methods of the invention.

[0066] Methods for obtaining an ATM gene are well known in the art,(see, e.g., Sambrook et al., 1989, supra). The DNA may be obtained bystandard procedures known in the art from cloned DNA (e.g., a DNA“library”), and preferably is obtained from a cDNA or genomic libraryprepared from tissues with high level expression of the protein, bychemical synthesis, by cDNA cloning, or by the cloning of genomic DNA,or fragments thereof, purified from the desired cell (see, e.g.,Sambrook et al., 1989, supra; Glover, D. M. (ed.), 1985, supra), or asdescribed in the ATM patents cited above. Whatever the source, the geneshould be molecularly cloned into a suitable vector for propagation ofthe gene.

[0067] Further selection can be carried out on the basis of theproperties of the gene, e.g., if the gene encodes a protein producthaving the isoelectric, electrophoretic, amino acid composition, partialor complete amino acid sequence, antibody binding activity, ligandbinding profile, particularly ATM kinase substrate binding specificity,or enzymatic activity of ATM protein as disclosed herein. Thus, thepresence of the gene may be detected by assays based on the physical,chemical, immunological, or functional properties of its expressedproduct.

[0068] The present invention also relates to assay and expressionsystems that employ cloning vectors containing genes encoding wild-typeATM, or analogs and derivatives of ATM that have the same or homologousfunctional activity as ATM. Thus, although ATM per se is a well-knownprotein, its expression and use in assays of the invention, such as thein vitro cell-free screening assay, is contemplated. The production anduse of derivatives and analogs related to ATM are within the scope ofthe present invention. In a specific embodiment, the derivative oranalog is functionally active, i.e., capable of exhibiting one or morefunctional activities associated with a full-length, wild-type ATM ofthe invention. Chimeric fusion proteins with ATM, such as FLAG, GST, orHIS-tagged ATM, are contemplated, as are fusion proteins that containfunctional domains (discussed below).

[0069] ATM derivatives can be made by altering encoding nucleic acidsequences by substitutions, additions or deletions that provide forfunctionally equivalent molecules. Preferably, derivatives are made thathave enhanced or increased functional activity relative to native ATM.In another embodiment, exemplified infra, a kinase dead derivative ofATM can be prepared. Preferably, the kinase dead derivative (or mutant)is dominant-negative.

[0070] Due to the degeneracy of nucleotide coding sequences, other DNAsequences which encode substantially the same amino acid sequence as anATM gene may be used in the practice of the present invention. Theseinclude but are not limited to allelic genes, sequence variants of ATM,and functional variants of ATM (see, infra).

[0071] The genes encoding ATM derivatives of the invention can beproduced by various methods known in the art. The sequence can becleaved at appropriate sites with restriction endonuclease(s), followedby further enzymatic modification if desired, isolated, and ligated invitro. In the production of the gene encoding a derivative of ATM, careshould be taken to ensure that the modified gene remains within the sametranslational reading frame as the ATM gene, uninterrupted bytranslational stop signals, in the gene region where the desiredactivity is encoded.

[0072] Additionally, the ATM-encoding nucleic acid sequence can bemutated in vitro or in vivo, to create and/or destroy translation,initiation, and/or termination sequences, or to create variations incoding regions and/or form new restriction endonuclease sites or destroypreexisting ones, to facilitate further in vitro modification. Anytechnique for mutagenesis known in the art can be used, including butnot limited to, in vitro site-directed mutagenesis (Hutchinson, C., etal., J. Biol. Chem. 253:6551, 1978; Zoller and Smith, DNA 3:479-488,1984; Oliphant et al., Gene 44:177, 1986; Hutchinson et al., Proc. Natl.Acad. Sci. U.S.A. 83:710, 1986), use of TAB linkers (Pharmacia), etc.PCR techniques are preferred for site directed mutagenesis (see Higuchi,1989, “Using PCR to Engineer DNA”, in PCR Technology: Principles andApplications for DNA Amplification, H. Erlich, ed., Stockton Press,Chapter 6, pp. 61-70).

[0073] The identified and isolated gene can then be inserted into anappropriate cloning vector. A large number of vector-host systems knownin the art may be used. Possible vectors include, but are not limitedto, plasmids or modified viruses, but the vector system must becompatible with the host cell used. Examples of vectors include, but arenot limited to, E. coli, bacteriophages such as lambda derivatives, orplasmids.

Optimization of Dominant-Negative ATM

[0074] Mutant forms or fragments of ATM can function asdominant-negative molecules when expressed exogenously (Morgan et al.,Mol. Cell Biol., 17:2020, 1997). However, an ATM fragment construct canbe quite unstable in its expression in human cells and prove difficultto use in many settings. One advantage of the invention is in providingATM constructs that inhibit wild-type ATM function in cells for studiesin both cell culture and animal models.

[0075] In each case, cDNA's after transfection or infection into cellsin culture are used to evaluate various aspects of ATM dysfunction,including assays for the G₂/M checkpoint, S-phase checkpoint andradiosensitivity (both MTT and clonogenic survival), as described morefully infra.

[0076] Full-Length Kinase-Dead Mutant.

[0077] A full-length mutant ATR expressed exogenously appears to havedominant-negative activity (Cliby et al., EMBO J, 17:159, 1998). Akinase-dead full length ATM mutant which can be stably expressed hasbeen generated (Example 1, infra; NCBI protein database accession no.1585222 for the amino acid sequence and accession no. U33841 for thecDNA sequence). In one embodiment, such a full-length dominant-negativeATM is prepared by mutating an amino acid corresponding to residueAsp-2870 of ATM, e.g., to Ala, or mutating an amino acid residuecorresponding to Asn-2875, e.g., to Lys, or preferably both. Both stabletransfectants and transient transfectants of various cell lines can bemade and tested for loss of ATM function by the variety of assaysdescribed herein.

[0078] ATM Fragments.

[0079] The full-length ATM cDNA is over 9 kb in length and is thus quitedifficult to manipulate. This large size also places significantrestrictions on vectors that can be used to introduce the cDNA, withparticular limitations on viral vectors. Thus, one further advantage ofthe invention is the discovery of a more reliable and facile way togenetically manipulate ATM function in tumor cells. In particular, someATM functions have been inhibited with a small fragment containing theleucine zipper region of ATM. Recombinant ATM chimeric fragmentexpression can be tested with a variety of available antibodies directedagainst ATM protein and/or with antibodies directed against an epitopetag, such as FLAG or GST, which can be placed on either the 5′ or 3′ endof the expressed sequence.

[0080] ATM fragments which may be tested include: (i) a smaller versionof FB2F, the leucine zipper region of ATM; (ii) the amino-terminal halfof the protein from translation start site through the middle of theprotein; (iii) the entire protein with just the kinase domain deleted;(iv) the carboxyl-terminal half of the protein. These fragments aretested for the ability to both complement and inhibit ATM function. If afragment complements (and appears to have kinase activity), then akinase-dead version of the fragment, which theoretically could act as adominant-negative peptide and is more easily manipulated and expressedthan full length ATM cDNA's, whether wild-type or mutant, is prepared.

[0081] ATM Kinase Substrate Recognition Consensus Sequence Motif(s)

[0082] An important breakthrough of the present invention isidentification of ATM kinase substrate recognition sequences, and aconsensus sequence motif for these sequences. Through use of reiterativesequence changes in fusion polypeptides used in in vitro kinase assays,a substrate motif has been defined for the ATM kinase. The basic motifrecognized by ATM is:

B₁-X-B₂-S-Q-X-X (SEQ ID NO:1)

[0083] where B₁ is a hydrophobic amino acid, B₂ is a hydrophobic aminoacid or aspartate, X is any amino acid, Q is glutamine, and S is serine(the amino acid which is phosphorylated by ATM). A number of differentsequences fitting this motif have been tested and two sequences appearto provide the optimal target sequence. These two sequences are:P-P-D-S-Q-E-X (SEQ ID NO:2) and L-P-[L or A]-S-Q-[D or P]-X (SEQ IDNOS:3-6)

[0084] where P is proline, D is aspartic acid, E is glutamic acid, L isleucine, A is alanine, and X is again any amino acid.

[0085] By analyzing proteins for the presence of a sequencecorresponding to the consensus sequence motif using sequence comparisonalgorithm, such as those mentioned below, it is possible to identifyproteins which are physiologic targets of the ATM kinase, termed herein“ATM target proteins”. A sequence corresponds to the motif when it hasthe defining amino acid residues in the appropriate positions relativeto each other. One known ATM target protein is p53. Once suchphysiological targets are identified, the cellular processes involved inphosphorylation (or inhibition of phosphorylation) by the ATM kinase canbe characterized. Identification of such processes permits one to screenfor and optimize small molecule inhibitors or inducers of ATM kinaseactivity to agonize or antagonize a particular cellular process.Clarification of these sequences also distinguishes ATM substratespecificity from other related kinases, such as ATR and DNA-PK. Theseclarifications aid in the development of modulators with enhancedspecificity for the ATM kinase. Summaries of the putative motifs foreach of these three kinases as defined by the in vitro kinase assays ofthe invention are shown in Table 4 in Example 3.

[0086] In a specific embodiment, a reiterative approach is used toidentify consensus putative target motifs for ATM and related kinases.Beginning with the amino terminal p53 target sequence containing serine15, which we have identified as an ATM target (Example 2), a plasmidfusing this peptide sequence in-frame with a GST sequence can begenerated and then expressed in bacteria. The GST-fusion polypeptide ispurified and then used as a target in the in vitro kinase assay usingATM, and optimally ATM-kinase dead, ATR, and/or DNA-PK_(CS) as thekinase. Selected mutations can be made in the p53 (or other) targetsequence to determine which mutations were capable of altering (eitherdecreasing or increasing) the ability of the kinase to phosphorylate theserine residue of interest.

[0087] Insights gained from this mutagenesis approach allow furtherdefinition of the putative target sequence for the ATM (and other)kinases. Searching sequence databases permits identification of proteinscontaining serine residues in sequences which are potentialphysiological targets of ATM. These sequences can be generated in theGST-plasmid (Example 1) and another GST-fusion polypeptide was thusavailable for testing (as demonstrated in Example 3). The results fromtesting such known sequences permit further refinement of the targetconsensus motif, which may then be used in a reiterative manner toidentify other potential physiologic target proteins of the ATM kinase.

[0088] As shown in Example 3, using the in vitro kinase assay andvarious peptide substrates to elucidate phosphorylation site motifs, wehave been able to screen protein databases for putative kinase targetsfor ATM. Candidate peptide sequences from these proteins were tested fortheir ability to be phosphorylated by ATM in the in vitro kinase assay.This accomplishes two goals: 1) further refinement of the ATM kinasesubstrate motif and, 2) identification of potential physiologic targetsof the ATM kinase, which can be tested for physiologic relevance inintact cells. The list of proteins and sites below represents theproteins, and sites within those proteins, which represent valid invitro targets for the ATM kinase. It should also be noted that thisapproach not only identifies protein targets, but it simultaneouslyprovides the invaluable information about which site in the protein isphosphorylated. Such information leads to rapid insights into thephysiological significance of phosphorylation events.

[0089] A set of proteins which contain sequences and are potential ATMtarget proteins identified using this approach are listed below (Table1). Subsequently, more quantitative experiments (Example 3) establishedthat some ATM substrate peptides are not highly phosphorylated. In apreferred aspect of the invention, proteins that contain putativesubstrate sequences that demonstrate at least about 20% of the level ofphosphorylation as the p53 peptide with S15 are considered goodcandidates for regulation or activation by ATM. However, phosphorylationof a putative substrate peptide indicates a high probability, but not acertainty, that the protein is a target of ATM phosphorylation in vivo.Thus, preferably, putative ATM phosphorylation site peptides show atleast about 20% of the phosphorylation of the p53 site containing S15.TABLE 1 Proteins Containing Sequences Phosphorylated by ATMPhosphorylation ATM Targets Activity Site Sequence SEQ ID NO: 53 (invivo) Tumor suppression SVEPPLSQETFSDL 7 NBS/p95 DNA NijmegenTPGPSLSQGVSVDE 10 Breakage syndrome MRE11 (SQ1) Double strand DNAQQLFYISQPGSSVV 11 break repair PHASI Insulin signaling EPPMEASQSHLRNS 9(obesity) CHK1 (SQ1) Cell cycle check point NVKYSSSQPEPRTG 16 WERNER(SQ1) Aging EKAYSSSQPVISAQ 21 CHK1 (SQ2) Cell cycle check pointVQGISFSQPTCPDH 17 PTS 1 Tumor suppression WETPDLSQAEIEQK 37 CUT1 Spindlecheck point GASPVLSQGVDPRS 36 ATM440 Autophosphorylation PLLMILSQLLPQQR24 BRCA1 (SQ2) Breast cancer DCSGLSSQSDILTT 33 RAD17 (SQ1) DNA damageresponses TWSLPLSQDSASEL 18 RAD17 (SQ2) DNA damage responsesASELPASQPQPFSA 19

[0090] As discussed below, several of these proteins are of greatphysiologic interest in and of themselves and even greater interest isgenerated by the fact that they may be physiologic targets of ATM.

[0091] Identification of protein sequences which do not appear to betargets of the ATM kinase has almost as much potential physiologicsignificance as identifying the proteins which are targets. For example,ATM plays a role in phosphorylating serine 15, but not serine 37, of p53after DNA damage, but both are thought to be important modifications ofthe protein after DNA damage. This approach allows us to conclude thatserine 37 is not a target of ATM, but is a reasonable target forDNA-PK_(CS). Similarly, we are able to conclude that the DNA repairenzyme, ligase IV, which contains a putative substrate sequence for anymember of this family of kinases, is phosphorylated by DNA-PK_(CS), butnot by ATM. Examples of proteins which have sequences which could serveas potential targets, but do not seem to be targets in our assays, arealso listed below (Table 2). TABLE 2 Examples of proteins whosesequences were not phosphorylated in vitro by ATM Peptide Sequence(putative Protein phosphorylation site) SEQ ID NO. TP1 SPLAPVSQQGWRSI 45CABL YPGIDLSQVYELLE 23 ATM(S2761) YKVVPLSQRSGVLE 25 ATR TVEPIISQLVTVLL22 P1-3K DLLMYLSQLVQALK 26 GEF RLRPLLSQLGGNSV 27 DNA POL-δLPCLEISQSVTGFG 30 E4F APEPPVSQELPCSR 31 BRCA2 KVSPYLSQFQQDKQ 29β-ADAPTIN CRAPEVSQHVYQAY 28 MRB11(SQ2) FSVLRFSQKFVDRV 12 MRE11(SQ3)RARALRSQSEESAS 13 MRE11(SQ4) SASRGGSQRGRAFK 14

[0092] BRCA2, another familial breast cancer gene product, appears to bephosphorylated by ATR, but not by ATM. The peptide sequence in BRCA2recognized by ATR is: KVSPYLSQFQQDKQ (SEQ ID NO:29). Another example ofdistinctions which can be drawn with this approach is that the relatedkinase, DNA-PK, can phosphorylate both serine 15 and 37 in p53 while ATMphosphorylates only serine 15.

[0093] Based on these data, and the kinase target motif sequences shownin Table 3, we were able to evaluate kinase specificity for substratesequences. TABLE 3 Kinase Target Motif Sequences KINASE N − 5 N − 4 N −3 N − 2 N − 1 N* N + 1 N + 2 N + 3 N + 4 ATM X X P/M/I/G/F/Y X D/I/A/S/LS Q X X X (B except D) “BEST — — P P D S Q E X — TARGET” — — L P L/A S QD/P X — DNA-PK V/L X P X L/P S/T Q E/A X F/D ATR X X L/P X L/A/V S/T Q XX X

[0094] ATM, a serine kinase, depends on hydrophobic residues immediatelyamino terminal to SQ at N−3 and N−1; it is unaffected by substitutionswhich are carboxyl terminal to N+1 or amino terminal to N−3.

[0095] DNA-PK, a serine-threonine kinase similar to ATM, but exhibitssome activity on threonine and depends on V at N−5 and E/A and F/D atN+2 and N+4 respectively.

[0096] ATR, a serine-threonine kinase which is similar to ATM, exhibitssome activity on threonine and is insensitive to changing N−1 in the p53amino terminal sequence to valine (this change abrogates ATM activity).

[0097] Of interest for the present invention is identification ofpolypeptide sequences that contain a putative ATM kinase substraterecognition sequence but cannot be phosphorylated. Such sequences canbind to the active site of ATM and competitively inhibit ATMphosphorylation of target proteins. Accordingly, such a sequence istermed herein a “competitive ATM kinase substrate recognition sequence”.In a specific embodiment, such sequences lack an amino acid residue thatis phosphorylated by ATM kinase, e.g., serine or, to a lesser extent,threonine.

ATM Target Proteins

[0098] Using the consensus motif, various proteins were identified bycomputer analysis as containing putative ATM kinase substraterecognition sites. A simple algorithm written to search the humanprotein database was used to identify sequences corresponding to apreliminary motif based on sequences from p53 and PHASI. These siteswere introduced into chimeric constructs (described infra) and testedfor phosphorylation by ATM. Based on this analysis, a number of ATMtarget proteins in addition to p53 have been identified.

[0099] Furthermore, allelic variants, degenerate coding sequences,truncated derivative ATM target proteins containing the ATM substraterecognition site, and ATM target protein derivatives with conservedamino acid substitutions (function conservative variants), and thenucleic acids encoding such variants and derivatives, can also beprepared. The function (where known) of these proteins, and the role ofATM in regulating the activity of these target proteins is discussedbelow:

[0100] p53 is the most commonly mutated gene in human cancer. It plays acritical role in helping cells respond to cytotoxic stresses, such asionizing irradiation, DNA base damage, hypoxia, etc. Serine 15, but notserine 37, is phosphorylated by ATM. This protein is important for bothtumor development and tumor responses to therapy.

[0101] NBS is the protein produced by the gene mutated in the “NijmegenBreakage Syndrome”. Children with this genetic disorder are similar tothose with Ataxia-telangiectasia (AT), except that they exhibitmicrocephaly and do not exhibit ataxia. NBS protein has recently beenshown to be part of a protein complex that forms at sites of doublestrand DNA breaks and appears to be critical for normal responses andrepair following DNA breakage. It is likely that ATM and NBS function inthe same pathway in helping cells respond to ionizing radiation. Thisobservation provides the first biochemical link between the two.

[0102] MRE11 is a part of the DNA double strand break repair complex(along with NBS), establishing a further link between ATM and DNA damageresponses. Identification of the sites which are phosphorylated in bothNBS and MRE11 focuses attention on these sites as having particularphysiologic relevance, and facilitates understanding their roles (byproviding potential sites within these proteins to mutate in functionalassays) and mechanisms of action in DNA damage response pathways.

[0103] PHASI is a member of the PHAS family that appears to be primarilyexpressed in adipocytes. PHAS proteins regulate protein translation inresponse to a number of stimuli, including insulin stimulation. The sitein PHASI identified as a putative ATM target is not present in the otherrelated PHAS proteins, suggesting that ATM activity is solely directedto PHASI in adipocytes. This observation may help explain the extremelythin nature of AT patients and potentially provides a novel mechanismfor obesity treatment as it relates to fat production.

[0104] CHK1 is a cell cycle checkpoint protein involved in controllingcell cycle progression following DNA damage. Proteins with this functionprobably play important roles in both cancer development and determiningcellular responses following exposure to DNA damaging agents, such asirradiation and chemotherapy. We have identified two putative ATM targetsites within CHK1; this will facilitate understanding the specific roles(by providing potential sites within CHK1 to mutate in functionalassays) and mechanisms of action of CHK1.

[0105] Werner's protein is a recently identified gene product which ismutated in Werner's Syndrome, a dramatic syndrome of premature aging.The protein appears to be a DNA helicase and identification of thisprotein as an ATM target facilitates understanding of both diseases andthe functions of both of these proteins.

[0106] PTS1 is our designation (“putative tumor suppressor”) for a genelocated on chromosome 3 on the ‘p’ arm at 3p21.3, which has not yet beennamed (GenBank accession number 3834393). The chromosomal location ofthe gene is a very common site of chromosomal loss in a variety ofcarcinomas. In a search for the important tumor suppressor gene onchromosome 3p21.3 in small cell lung cancer, this gene was identifiedand the complete DNA sequence of this gene was recently submitted to thesequence databases. Using the “reiterative” approach detailed herein,this peptide sequence proved to be an excellent in vitro target of theATM protein kinase. Subsequent data with a fragment of this protein ofabout 100 amino acids confirm this observation. The discovery that thisprotein, which may be the long sought after ‘3p’ tumor suppressor, is atarget of ATM kinase provides insights that could greatly facilitateunderstanding the role and mechanism of action of the protein, which maybe important.

[0107] CUT1 is a protein which appears to be involved in the formationof mitotic spindles and thus plays a critical role in cell cycleprogression. Little is known about its mechanism of action, butidentification of the ATM phosphorylation site provides forinvestigating mechanistic questions. Since mitotic checkpoints havepotential relevance as targets for cancer treatments, this may turn outto have therapeutic relevance as well as importance for general cellbiology.

[0108] ATM is a phosphoprotein, and our data suggest that it can beautophosphorylated. Examining the sequence of ATM uncovered a number ofpotential sites, and one peptide sequence has been identified asautophosphorylated at amino acid residue 440 by ATM. This discoveryprovides novel insights into regulation of ATM function and may be usedto inhibit ATM function in selected pathways, such as specifically inradiation-induced responses, without altering other aspects of ATMfunction. This has particular relevance in the optimal development of ainhibitor of ATM to be used for radiosensitization clinically.

[0109] BRCA1 is a gene which has been identified as an importantfamilial breast cancer susceptibility gene. The protein product of BRCA1has been implicated in DNA damage response pathways, but there is stilla poor level of understanding of BRCA1 function. Identification of thisprotein as an ATM target provides insights into breast cancerdevelopment and treatment and into general aspects of radiation biologyranging from tumor development to tumor response to therapy.

[0110] hRAD17 is the human homolog of a gene product which is a criticalcell cycle checkpoint protein in yeast. Its relevance to human diseaseis not known, but identification of this protein as a target of ATMprovides important insights into the understanding of radiationresponses and has implications for tumor responses to therapy. Thisobservation has the potential to lead to the identification of othertargets for enhancing the radiosensitivity of human tumors.

ATM Substrate Polypeptides

[0111] The present invention further provides chimeric proteins thatcontain putative ATM kinase substrate recognition sequences (termedherein “ATM substrate polypeptides”), including nucleic acids encodingsuch ATM substrate polypeptides, and vectors for expression of such ATMsubstrate polypeptides for in vitro (e.g., cell free), ex vivo (e.g.,cell line-based), or in vivo assays. Because the present inventionprovides the sequence recognized by ATM for phosphorylation, in apreferred embodiment, fusion polypeptides comprising a putative or knownATM kinase recognition sequence from a protein can be prepared andtested for binding to or phosphorylation by ATM, e.g., in a cell-free invitro assay as exemplified in Example 1. Alternatively, ATM kinasetarget proteins discussed in greater detail in the section above canalso be used in various assays for ATM kinase enzymatic (i.e.,phosphorylation) activity.

[0112] ATM substrate polypeptides can be obtained using the molecularbiological techniques described in connection with ATM, supra.

[0113] As used herein, the term “fusion polypeptide” refers to achimeric construct comprising a structural portion and an ATM kinasesubstrate recognition sequence portion. The term “structural portion”refers to a part of the fusion polypeptide that provides a generalizedfunction, such as specific binding, reporter enzymatic activity, or thatliterally supplies sufficient secondary, tertiary, and/or quaternarystructure so that ATM kinase can bind to and phosphorylate the substraterecognition sequence portion. Examples of structural portions include,but are not limited to, FLAG, GST, and HIS-tag. FLAG and GST are readilyrecognized by antibodies, and permit immunoseparation, e.g.,immunoprecipitation or detection by immunoassay (similar epitope tagscan be included in recombinant ATM, as described above, or recombinantATM target proteins). A HIS-tag permits chromatographic separation on anickel (Ni)-chelation column. Examples of structural portions withreporter activity include β-galactosidase, chloramphenicol transferase,horseradish peroxidase, alkaline phosphatase, luciferase, and greenfluorescent protein. Other possible structural portions include antibodyFc portions, targeting molecules such as hormones or transferrin, or anyother polypeptide that has a desired sequence. Preferably the structuralportion does not itself contain a sequence that is phosphorylated by ATMkinase, i.e., it lacks an ATM kinase substrate recognition sequence.This ensures that only the putative substrate recognition sequenceportion of the fusion polypeptide will be phosphorylated (if at all).

[0114] The term “ATM kinase substrate recognition sequence” refers to asequence that has (termed a known recognition sequence) or appears tohave (termed a putative recognition sequence) an ATM kinase substraterecognition sequence, i.e., it has a sequence corresponding to the ATMkinase substrate recognition consensus sequence motif. As exemplifiedinfra, any putative ATM kinase substrate recognition sequence portioncan be joined with the structural portion and tested for recognition andphosphorylation by ATM. Using chimeric constructs of the invention, anumber of ATM kinase substrate recognition sequences have beenidentified; other sequences containing serine have been eliminated asnot phosphorylated by ATM. In a specific embodiment, the sequencephosphorylated by ATM is selected from the following group:SVEPPLSQETFSDL (SEQ ID NO:7); TPGPSLSQGVSVDE (SEQ ID NO:10);QQLFYISQPGSSVV (SEQ ID NO:11); EPPMEASQSHLRNS (SEQ ID NO:9);NVKYSSSQPEPRTG (SEQ ID NO:16); EKAYSSSQPVISAQ (SEQ ID NO:21);VQGISFSQPTCPDH (SEQ ID NO:17); WETPDLSQAEIEQ (SEQ ID NO:37);GASPVLSQGVDPR (SEQ ID NO:36); PLLMILSQLLPQQR (SEQ ID NO:24);DCSGLSSQSDILTT (SEQ ID NO:33); TWSLPLSQDSASEL (SEQ ID NO:18); andASELPASQPQPFSA (SEQ ID NO:19).

[0115] In a further embodiment, a substrate recognition sequence in thefusion polypeptide can bind ATM without being phosphorylated, e.g., acompetitive ATM kinase substrate recognition sequence. Such competitivesequences can be identified using the binding and phosphorylation assaysof the invention.

[0116] To assay for phosphorylation activity, ATM kinase is contactedwith a fusion polypeptide and phosphorylation of the fusion polypeptideis detected. This approach has, for example, permitted refinement of theconsensus sequence motif by establishing whether a putative recognitionsequence is phosphorylated by ATM kinase.

[0117] Binding activity of a fusion polypeptide with ATM can be tested,e.g., by a competitive assay, immunoassay, or other standard method inthe art. The physiological relevance of phosphorylation of the fusionpolypeptide can be confirmed by testing with the target protein fromwhich the recognition sequence was obtained; further analysis caninvolve evaluating the effects on cellular processes, as described ingreater detail infra.

[0118] Furthermore, the substrate recognition sequence of a targetprotein can be systematically mutated in a fusion polypeptide to furtherexplore the sequence specificity of ATM kinase, as exemplified infra.

[0119] The fusion polypeptides can also be used to identify an optimaltarget sequence for ATM, either through mutagenesis or by testingsequences from putative target proteins. An optimized ATM kinasesubstrate recognition sequence may be distinguished from other kinaserecognition sequences, as exemplified, infra.

[0120] A fusion polypeptide comprising an ATM kinase substraterecognition sequence fused to a non-target, or structural, amino acidsequence can be produced by any means. In one embodiment, such a fusionpolypeptide is produced by recombinant expression of a nucleic acidencoding the protein (comprising an ATM kinase substraterecognition-coding sequence joined in-frame to a structural codingsequence). The nucleic acid can be made by ligating the appropriatenucleic acid sequences encoding the desired amino acid sequences to eachother by methods known in the art, which are described herein, in theproper reading frame, and expressing the chimeric product by methodscommonly known in the art. Chimeric genes comprising putative ATM kinasesubstrate recognition sequence portions fused to any heterologousprotein-encoding sequences may be constructed. Alternatively, it ispossible to prepare such a fusion polypeptide by synthetic orsemi-synthetic methods, including solid phase peptide synthesis orpeptide condensation synthesis, or a combination thereof.

Crystal Structure of ATM Kinase

[0121] Knowledge of the ATM kinase substrate recognition sequence canprovide a theoretical basis for predicting the structure of potentialmodulations, such as agonists or antagonists, of the protein.Identification and screening of modulators is further facilitated bydetermining structural features of the recognition sequence, e.g., usingX-ray crystallography, neutron diffraction, nuclear magnetic resonancespectrometry, and other techniques for structure determination. Thesetechniques provide for the rational design or identification of agonistsand antagonists of ATM kinase.

[0122] In particular, when designing or optimizing small moleculeinhibitors, it is extremely helpful to have a crystal structure of thetarget molecule. This is a particular challenge with ATM because of itsenormous size (approximately 370 kd peptide). Crystallization may beperformed either with the full length protein or with an expressedfragment containing the kinase domain. At this time, it appears that the“Rad3” fragment in the C-terminal half of the protein is expressed atreasonable levels in mammalian cells. Full-length ATM has beensuccessfully expressed in baculovirus. Thus, either full-length ATM orRad3 ATM can be used for crystallization studies. Initialcharacterizations of either ATM or “Rad3” proteins includes limitedproteolysis and domain analysis. This may aid in identifying an optimalpeptide to use for crystallization.

[0123] In a preferred embodiment, a composition comprising ATM and anATM substrate polypeptide (containing an ATM kinase substraterecognition site) are provided for co-crystallization (or otherstructure-function studies). The polypeptide can be a peptide having therecognition sequence, an ATM substrate polypeptide, or a fragment of anATM target protein containing the recognition sequence. In specificembodiments, one of the sequences disclosed herein is employed. Inanother embodiment, co-crystallization is performed in a manganese-freesolution to prevent phosphorylation and discharge of the substratepolypeptide.

Recombinant Expression Systems

[0124] The present invention contemplates various cloning and expressionvectors for expression of the proteins and polypeptides describedherein, including without limitation ATM kinase, ATM kinase-dead mutantsand other ATM derivatives, target proteins, mutated target proteins, ATMsubstrate polypeptides, and the like. Such expression vectors can beused to transform cells in vivo or in vitro for ATM kinase activityassays, or the investigates the role of ATM or cellular processes.Furthermore, recombinant expression systems can be used to produce ATMand target proteins or substrate polypeptides for extracellular activityand binding assays. The molecular biological techniques described abovecan be used to prepare expression systems of the invention.

[0125] A wide variety of host/expression vector combinations (i.e.,expression systems) may be employed in expressing the DNA sequences ofthis invention. Useful expression vectors, for example, may consist ofsegments of chromosomal, non-chromosomal and synthetic DNA sequences.Suitable vectors include derivatives of SV40 and known bacterialplasmids, e.g., E. coli plasmids col El, pCR1, pBR322, pMal-C2, pET,pGEX (Smith et al., Gene 67:31-40, 1988), pMB9 and their derivatives,plasmids such as RP4; phage DNAS, e.g., the numerous derivatives ofphage 1, e.g., NM989, and other phage DNA, e.g., M13 and filamentoussingle stranded phage DNA; yeast plasmids such as the 2m plasmid orderivatives thereof; vectors useful in eukaryotic cells, such as vectorsuseful in insect or mammalian cells; vectors derived from combinationsof plasmids and phage DNAs, such as plasmids that have been modified toemploy phage DNA or other expression control sequences; and the like. Anexample of a mammalian cell expression system of the invention includes,but is by no means limited to, 293T cells, as exemplified infra. Inaddition, various tumor cells lines can be used in expression systems ofthe invention.

[0126] Expression of the protein or polypeptide may be controlled by anypromoter/enhancer element known in the art, but these regulatoryelements must be functional in the host selected for expression.Promoters which may be used to control gene expression include, but arenot limited to, cytomegalovirus (CMV) promoter, the SV40 early promoterregion (Benoist and Chambon, 1981, Nature 290:304-310), the promotercontained in the 3′ long terminal repeat of Rous sarcoma virus(Yamamoto, et al., Cell 22:787-797, 1980), the herpes thymidine kinasepromoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445,1981), the regulatory sequences of the metallothionein gene (Brinster etal., Nature 296:39-42, 1982);

[0127] prokaryotic expression vectors such as the β-lactamase promoter(Villa-Komaroff, et al., Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731,1978), or the tac promoter (DeBoer, et al., Proc. Natl. Acad. Sci.U.S.A. 80:21-25, 1983); see also “Useful proteins from recombinantbacteria” in Scientific American, 242:74-94, 1980; promoter elementsfrom yeast or other fungi such as the Gal 4 promoter, the ADC (alcoholdehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkalinephosphatase promoter.

Expression Vectors

[0128] Preferred vectors, particularly for cellular assays in vitro andanimal models or therapeutics in vivo or ex vivo, are viral vectors,such as lentiviruses, retroviruses, herpes viruses, adenoviruses,adeno-associated viruses, vaccinia viruses, baculoviruses, and otherrecombinant viruses with desirable cellular tropism. Thus, a geneencoding a functional or mutant protein or polypeptide can be introducedin vivo, ex vivo, or in vitro using a viral vector or through directintroduction of DNA. Expression in targeted tissues can be effected bytargeting the transgenic vector to specific cells, such as with a viralvector or a receptor ligand, or by using a tissue-specific promoter, orboth. Targeted gene delivery is described in International PatentPublication WO 95/28494, published October 1995.

[0129] Viral vectors commonly used for in vivo or ex vivo targeting andtherapy procedures are DNA-based vectors and retroviral vectors. Methodsfor constructing and using viral vectors are known in the art (see,e.g., Miller and Rosman, BioTechniques, 7:980-990, 1992). Preferably,the viral vectors are replication defective, that is, they are unable toreplicate autonomously in the target cell. Preferably, the replicationdefective virus is a minimal virus, i.e., it retains only the sequencesof its genome which are necessary for encapsidating the genome toproduce viral particles.

[0130] DNA viral vectors include an attenuated or defective DNA virus,such as but not limited to herpes simplex virus (HSV), papillomavirus,Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), andthe like. Defective viruses, which entirely or almost entirely lackviral genes, are preferred. Defective virus is not infective afterintroduction into a cell. Use of defective viral vectors allows foradministration to cells in a specific, localized area, without concernthat the vector can infect other cells. Thus, a specific tissue can bespecifically targeted. Examples of particular vectors include, but arenot limited to, a defective herpes virus 1 (HSV 1) vector (Kaplitt etal., Molec. Cell. Neurosci. 2:320-330, 1991; International PatentPublication No. WO 94/21807, published Sept. 29, 1994; InternationalPatent Publication No. WO 92/05263, published Apr. 2, 1994); anattenuated adenovirus vector, such as the vector described byStratford-Perricaudet et al. (J. Clin. Invest. 90:626-630, 1992; seealso La Salle et al., Science 259:988-990, 1993); and a defectiveadeno-associated virus vector (Samulski et al., J. Virol. 61:3096-3101,1987; Samulski et al., J. Virol. 63:3822-3828, 1989; Lebkowski et al.,Mol. Cell. Biol. 8:3988-3996, 1988).

[0131] Various companies produce viral vectors commercially, includingbut by no means limited to Avigen, Inc. (Alameda, Calif.; AAV vectors),Cell Genesys (Foster City, Calif.; retroviral, adenoviral, AAV vectors,and lentiviral vectors), Clontech (retroviral and baculoviral vectors),Genovo, Inc. (Sharon Hill, Pa.; adenoviral and AAV vectors), Genvec(adenoviral vectors), IntroGene (Leiden, Netherlands; adenoviralvectors), Molecular Medicine (retroviral, adenoviral, AAV, and herpesviral vectors), Norgen (adenoviral vectors), Oxford BioMedica (Oxford,United Kingdom; lentiviral vectors), and Transgene (Strasbourg, France;adenoviral, vaccinia, retroviral, and lentiviral vectors).

[0132] Preferably, for in vivo administration, an appropriateimmunosuppressive treatment is employed in conjunction with the viralvector, e.g., adenovirus vector, to avoid immuno-deactivation of theviral vector and transfected cells. For example, immunosuppressivecytokines, such as interleukin-12 (IL-12), interferon-γ (IFN-γ), oranti-CD4 antibody, can be administered to block humoral or cellularimmune responses to the viral vectors (see, e.g., Wilson, NatureMedicine, 1995). In that regard, it is advantageous to employ a viralvector that is engineered to express a minimal number of antigens.

[0133] Adenovirus Vectors.

[0134] Adenoviruses are eukaryotic DNA viruses that can be modified toefficiently deliver a nucleic acid of the invention to a variety of celltypes. Various serotypes of adenovirus exist. Of these serotypes,preference is given, within the scope of the present invention, to usingtype 2 or type 5 human adenoviruses (Ad 2 or Ad 5) or adenoviruses ofanimal origin (see WO94/26914). Those adenoviruses of animal originwhich can be used within the scope of the present invention includeadenoviruses of canine, bovine, murine (example: Mav1, Beard et al.,Virology 75 (1990) 81), ovine, porcine, avian, and simian (example: SAV)origin. Preferably, the adenovirus of animal origin is a canineadenovirus, more preferably a CAV2 adenovirus (e.g., Manhattan or A26/61strain (ATCC VR-800), for example). Various replication defectiveadenovirus and minimum adenovirus vectors have been described(WO94/26914, WO95/02697, WO94/28938, WO94/28152, WO94/12649, WO95/02697WO96/22378). The replication defective recombinant adenovirusesaccording to the invention can be prepared by any technique known to theperson skilled in the art (Levrero et al., Gene 101:195 1991; EP 185573; Graham, EMBO J. 3:2917, 1984; Graham et al., J. Gen. Virol. 36:591977). Recombinant adenoviruses are recovered and purified usingstandard molecular biological techniques, which are well known to one ofordinary skill in the art.

[0135] Adeno-Associated Viruses.

[0136] The adeno-associated viruses (AAV) are DNA viruses of relativelysmall size which can integrate, in a stable and site-specific manner,into the genome of the cells which they infect. They are able to infecta wide spectrum of cells without inducing any effects on cellulargrowth, morphology or differentiation, and they do not appear to beinvolved in human pathologies. The AAV genome has been cloned, sequencedand characterized. The use of vectors derived from the AAVs fortransferring genes in vitro and in vivo has been described (see WO91/18088; WO 93/09239; U.S. Pat. Nos. 4,797,368, 5,139,941, EP 488 528).The replication defective recombinant AAVs according to the inventioncan be prepared by cotransfecting a plasmid containing the nucleic acidsequence of interest flanked by two AAV inverted terminal repeat (ITR)regions, and a plasmid carrying the AAV encapsidation genes (rep and capgenes), into a cell line which is infected with a human helper virus(for example an adenovirus). The AAV recombinants which are produced arethen purified by standard techniques.

[0137] Retrovirus Vectors.

[0138] In another embodiment the gene can be introduced in a retroviralvector, e.g., as described in Anderson et al., U.S. Pat. No. 5,399,346;Mann et al., 1983, Cell 33:153; Temin et al., U.S. Pat. No. 4,650,764;Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., 1988, J. Virol.62:1120; Temin et al., U.S. Pat. No. 5,124,263; EP 453242, EP178220;Bernstein et al. Genet. Eng. 7 (1985) 235; McCormick, BioTechnology 3(1985) 689; International Patent Publication No. WO 95/07358, publishedMar. 16, 1995, by Dougherty et al.; and Kuo et al., 1993, Blood 82:845.The retroviruses are integrating viruses which infect dividing cells.The retrovirus genome includes two LTRs, an encapsidation sequence andthree coding regions (gag, pol and env). In recombinant retroviralvectors, the gag, pol and env genes are generally deleted, in whole orin part, and replaced with a heterologous nucleic acid sequence ofinterest. These vectors can be constructed from different types ofretrovirus, such as, HIV, MoMuLV (“murine Moloney leukaemia virus” MSV(“murine Moloney sarcoma virus”), HaSV (“Harvey sarcoma virus”); SNV(“spleen necrosis virus”); RSV (“Rous sarcoma virus”) and Friend virus.Suitable packaging cell lines have been described in the prior art, inparticular the cell line PA317 (U.S. Pat. No. 4,861,719); the PsiCRIPcell line (WO 90/02806) and the GP+envAm-12 cell line (WO 89/07150). Inaddition, the recombinant retroviral vectors can contain modificationswithin the LTRs for suppressing transcriptional activity as well asextensive encapsidation sequences which may include a part of the gaggene (Bender et al., J. Virol. 61:1639, 1987). Recombinant retroviralvectors are purified by standard techniques known to those havingordinary skill in the art.

[0139] Retrovirus vectors can also be introduced by recombinant DNAviruses, which permits one cycle of retroviral replication and amplifiestranfection efficiency (see WO 95/22617, WO 95/26411, WO 96/39036, WO97/19182).

[0140] Lentivirus Vectors.

[0141] In another embodiment, lentiviral vectors are can be used asagents for the direct delivery and sustained expression of a transgenein several tissue types, including brain, retina, muscle, liver andblood. The vectors can efficiently transduce dividing and nondividingcells in these tissues, and maintain long-term expression of the gene ofinterest. For a review, see, Naldini, Curr. Opin. Biotechnol., 9:457-63,1998; see also Zufferey, et al., J. Virol., 72:9873-80, 1998).Lentiviral packaging cell lines are available and known generally in theart. They facilitate the production of high-titer lentivirus vectors forgene therapy. An example is a tetracycline-inducible VSV-G pseudotypedlentivirus packaging cell line which can generate virusparticles attiters greater than 106 IU/ml for at least 3 to 4 days (Kafri, et al.,J. Virol., 73: 576-584, 1999). The vector produced by the inducible cellline can be concentrated as needed for efficiently transducingnondividing cells in vitro and in vivo.

[0142] Non-Viral Vectors.

[0143] In another embodiment, the vector can be introduced in vivo bylipofection, as naked DNA, or with other transfection facilitatingagents (peptides, polymers, etc.). Synthetic cationic lipids can be usedto prepare liposomes for in vivo transfection of a gene encoding amarker (Felgner, et. al., Proc. Natl. Acad. Sci. U.S.A. 84:7413-7417,1987; Felgner and Ringold, Science 337:387-388, 1989; see Mackey, etal., Proc. Natl. Acad. Sci. U.S.A. 85:8027-8031, 1988; Ulmer et al.,Science 259:1745-1748, 1993). Useful lipid compounds and compositionsfor transfer of nucleic acids are described in International PatentPublications WO95/18863 and WO96/17823, and in U.S. Pat. No. 5,459,127.Lipids may be chemically coupled to other molecules for the purpose oftargeting (see Mackey, et. al., supra). Targeted peptides, e.g.,hormones or neurotransmitters, and proteins such as antibodies, ornon-peptide molecules could be coupled to liposomes chemically.

[0144] Other molecules are also useful for facilitating transfection ofa nucleic acid in vivo, such as a cationic oligopeptide (e.g.,International Patent Publication WO95/21931), peptides derived from DNAbinding proteins (e.g., International Patent Publication WO96/25508), ora cationic polymer (e.g., International Patent Publication WO95/21931).

[0145] It is also possible to introduce the vector in vivo as a nakedDNA plasmid. Naked DNA vectors for gene therapy can be introduced intothe desired host cells by methods known in the art, e.g.,electroporation, microinjection, cell fusion, DEAE dextran, calciumphosphate precipitation, use of a gene gun, or use of a DNA vectortransporter (see, e.g., Wu et al., J. Biol.

[0146] Chem. 267:963-967, 1992; Wu and Wu, J. Biol. Chem.263:14621-14624, 1988; Hartmut et al., Canadian Patent Application No.2,012,311, filed Mar. 15, 1990; Williams et al., Proc. Natl. Acad. Sci.USA 88:2726-2730, 1991). Receptor-mediated DNA delivery approaches canalso be used (Curiel et al., Hum. Gene Ther. 3:147-154, 1992; Wu and Wu,J. Biol. Chem. 262:4429-4432, 1987). U.S. Pat. Nos. 5,580,859 and5,589,466 disclose delivery of exogenous DNA sequences, free oftransfection facilitating agents, in a mammal. Recently, a relativelylow voltage, high efficiency in vivo DNA transfer technique, termedelectrotransfer, has been described (Mir et al., C.P. Acad. Sci.,321:893, 1998; WO 99/01157; WO 99/01158; WO 99/01175).

Screening Assays

[0147] Based on the present invention, a program to screen compounds orlibraries of compounds for their ability to modulate phosphorylation byATM kinase can be implemented. Modulation of ATM activity includesincreasing (agonizing) or inhibiting (antagonizing) ATM binding to ATMsubstrate polypeptides or ATM target proteins, or ATM-mediatedphosphorylation of substrate polypeptides or target proteins. Usingeither full-length ATM or fragments containing the kinase domain,compounds which modulate ATM kinase activity can be screened. Afterinitial identification and preliminary characterizations, such candidatemodulator compounds may be evaluated in cell-based and animal modelassays in order to determine their ability to function as modulators ofATM function (described in the following section).

[0148] Any screening technique known in the art can be used to screenfor ATM agonists or antagonists. For example, various binding assays forATM binding to polypeptides that comprise an ATM kinase substraterecognition sequence or a competitive kinase substrate recognitionsequence can be employed.

[0149] In general, screening for a compound that modulates ATM-mediatedphosphorylation involves detecting whether there is a change in thelevel of ATM-mediated phosphorylation of an ATM substrate polypeptide ora novel ATM target protein in the presence of a candidate compound,e.g., using the cell-free in vitro assay described above and inExample 1. An increase in the level of phosphorylation indicates thatthe compound agonizes ATM-mediated phosphorylation, and a decrease inthe level of phosphorylation indicates that the compound antagonizesATM-mediated phosphorylation. Preferably, the compound selectivelymodulates ATM-mediated phosphorylation, i.e., without affecting otherkinases, such as ATR or DNA-PK.

[0150] In a further embodiment, the screen can provide for detectinginhibition of a cellular process mediated by ATM phosphorylation of atarget protein, wherein inhibition of the activity is indicative ofinhibition of ATM as described in Example 4. For example, the cellularprocess may be loss of S-phase checkpoint, a defect in the G₂/Mcheckpoint, an increase in radiosensitivity, or increase in sensitivityto chemotherapeutic agents. Preferably, particularly for primaryscreening, the cellular process involves an novel ATM target protein.

[0151] In still another embodiment, the screen provides for detecting acompound that induces an ATM-regulated process in a cell, comprisingcontacting the cell with a candidate compound, and detecting whether theATM-mediated process is induced in the cell. Preferably, the cell isdefective for expression of ATM, or is modified to express adominant-negative ATM mutant.

[0152] In another embodiment, various reporter gene assays can be usedto evaluate changes in gene expression as a result of modulation of ATMactivity by a test compound. Preferably, the reporter gene expression istied to ATM-mediated phosphorylation. This can be accomplished byintroducing an ATM kinase substrate recognition sequence into a signaltransduction protein upstream of the reporter gene, or by inserting areporter gene into a gene whose expression is induced (or suppressed) inan ATM-regulated fashion. In a preferred embodiment, reporter geneexpression is controlled by an ATM target protein, especially a noveltarget protein as described herein. Reporter genes include greenfluorescent protein (GFP), luciferase, β-galactosidase (β-gal or lac-Z),chloramphenicol transferase (CAT), horseradish peroxidase, and alkalinephosphatase. In addition, expression levels of almost any protein can bedetected using a specific antibody.

[0153] As used herein, the term “compound” refers to any molecule orcomplex of more than one molecule that affects ATM function. The presentinvention contemplates screens for synthetic small molecule agents,chemical compounds, chemical complexes, and salts thereof as well asscreens for natural products, such as plant extracts or materialsobtained from fermentation broths. Other molecules that can beidentified using the screens of the invention include proteins andpeptide fragments, peptides, nucleic acids and oligonucleotides(particularly triple-helix-forming oligonucleotides), carbohydrates,phospholipids and other lipid derivatives, steroids and steroidderivatives, prostaglandins and related arachadonic acid derivatives,etc.

[0154] One approach to identifying such a compound uses recombinantbacteriophage to produce large libraries. Using the “phage method”(Scott and Smith, Science 249:386-390, 1990; Cwirla, et al., Proc. Natl.Acad. Sci., 87:6378-6382, 1990; Devlin et al., Science, 49:404-406,1990), very large libraries can be constructed (10⁶-10⁸ chemicalentities). A second approach uses primarily chemical methods, of whichthe Geysen method (Geysen et al., Molecular Immunology 23:709-715, 1986;Geysen et al. J. Immunologic Method 102:259-274, 1987; and the method ofFodor et al. (Science 251:767-773, 1991) are examples. Furka et al.(14th International Congress of Biochemistry, Volume #5, AbstractFR:013, 1988; Furka, Int. J. Peptide Protein Res. 37:487-493, 1991),Houghton (U.S. Pat. No. 4,631,211, issued December 1986) and Rutter etaL (U.S. Pat. No. 5,010,175, issued Apr. 23, 1991) describe methods toproduce a mixture of peptides that can be tested as agonists orantagonists. In another aspect, synthetic solid phase combinatoriallibraries (Needels et al., Proc. Natl. Acad. Sci. USA 90:10700-4, 1993;Ohlmeyer et al., Proc. Natl. Acad. Sci. USA 90:10922-10926, 1993; Lam etal., International Patent Publication No. WO 92/00252; Kocis et al.,International Patent Publication No. WO 9428028) and the like can beused to screen for ATM ligands according to the present invention.

Assays For Modulation of ATM Function

[0155] Screening for ATM function and modulation of ATM functioninvolves cell-based or animal model-based assays. Such assays can beused as secondary screens for the activity of candidate compoundsselected in a primary screen, e.g., the cell-free kinase assay describedabove and in Example 1. Furthermore, the assays for candidate ATMinhibitor compounds (antagonists) can be validated with a positivecontrol—the dominant-negative ATM mutant. Certain assays of theinvention provide improvements over conventional assays, and the presentinvention contemplates such improved cell-based assays or ATM functionas falling within its scope. Moreover, either an ATM inhibitordiscovered herein, or a dominant-negative mutant, can be used toestablish ATM-regulated pathways in cells. These models are also usefulto evaluate the effects of ATM inhibition on the function of othertreatments, such as radiation and chemotherapy.

Cell-Based Assays

[0156] S-Phase Checkpoint.

[0157] Loss of the S-phase checkpoint is one of the pathognomonicfeatures of cells lacking ATM function. This is a more difficultcheckpoint to assess than the G₁/S checkpoint and, in the past, requireda relatively cumbersome assay of incorporation of ³H-thymidine at veryearly time points after irradiation. The present invention optimizesaspects of this assay to enhance its reliability and ease of use.Furthermore, a sensitive and reliable non-radioactive, flow cytometricassay using incorporation of the thymidine analog, BrdUrd, at early timepoints after irradiation can be implemented.

[0158] G₂/M Checkpoint.

[0159] The defect in the G₂/M checkpoint in AT cells is an unusual one.Only cells which are in G₂ at the time of irradiation fail to arrest inG₂ and enter mitosis (normal cells will not enter mitosis) in thissetting. AT cells which are in S-phase at the time of irradiation willarrest when they get into G₂. In a typical cell cycle analysis usingflow cytometry, the increase in the number of G₂ cells after irradiationis actually a result of irradiated S-phase cells accumulating in G₂, andit takes at least a few hours to begin to see this effect. In order toassess the defect in the G₂/M checkpoint in AT cells, it has beennecessary to actually do mitotic spreads on cells at early time pointsafter irradiation and count the number of mitotic cells at various timesafter irradiation (Morgan et al., Mol. Cell. Biol., 17:2020, 1997). Insuch a scenario, after 30 minutes, there will be a decrease in thenumber of mitotic figures if cells arrest in G₂ (normal cells) and therewill be little to no decrease in mitotic figures at these time pointsafter irradiation in cells defective in the G₂/M checkpoint (e.g., ATcells).

[0160] A more rapid, objective quantitative assay for this G₂/Mcheckpoint as another assessment of ATM function in cells is provided.Two-color flow cytometry using a combination of PI may be used to assessDNA content and a mitotic specific antibody conjugated to FITC. Forexample, an antibody to phosphorylated Histone 113 (which isphosphorylated only in mitosis) and antibodies specific forphosphorylated MPM2 (which is also phosphorylated only in mitosis) canbe used to assess mitotic state of cells. The ability to distinguish theG₂ cells (4N content DNA, H-P negative) and M cells (4N content DNA,H3-P positive) allows accurate quantization of the number of G₂ and Mcells and easy assessment of the ability of G₂ cells to arrest prior toM at these early time points. This assay can serve as a facile way totest inhibitors for their ability to inhibit ATM function.

[0161] Radiosensitivity.

[0162] Another classic feature of loss of ATM function is enhancedradiosensitivity. The standard for assessing radiosensitivity hastypically been clonogenic survival assays. However, this is a long andsomewhat cumbersome assay. The present invention provides forutilization of MTT assays for a rapid assessment of increasedradiosensitivity caused by ATM dysfunction. An optimized MTT assay maybe used to quickly screen compounds for their ability to increasecellular radiosensitivity and promising compounds can be checked withstandard clonogenic survival assays.

[0163] Sensitivity to Agents Other Than Ionizing Radiation.

[0164] AT cells have also been reported to exhibit increased sensitivityto radiomimetic drugs, like bleomycin. Though the concomitant use of asystemic drug along with an ATM inhibitor would be difficult because ofincreased toxicity to normal tissues, such a drug conjugated to amolecule which targets the drug to the tumor could potentially be usedsuccessfully in combination with ATM inhibition. For example,conjugating a drug to an antibody directed against a ganglioside, suchas GD2, would target that drug to neuroblastoma cells. Concurrent use ofan ATM inhibitor may significantly enhance the sensitivity of the tumorcell to the tumor-directed drug. Similarly, an antibody against Her2-neucould be used in such a combination to more effectively treat a subsetof breast cancers. This is similar to the use of brachytherapy (seebelow), but uses a drug conjugated to the antibody or other targetingmolecule rather than a radioisotope. This concept is further developedbelow.

[0165] Candidate compounds can be tested for enhanced sensitivity in ATcells. One example of compounds for testing are the calicheamicins,which have been reported to be more toxic in AT cells (Sullivan andLyne, Mut. Res., 245:171, 1990) and have been successfully conjugated toan antibody in the treatment of neuroblastoma cells (Lode et al., CancerRes., 58:2925, 1998). Other compounds can be similarly tested andcandidate compounds can be tested in the model animal systems as well.

[0166] Transfectants.

[0167] A variety of tumor cell lines can be transfected both stably andtransiently and tested in the various ATM functional assays. These celllines include: H1299; MEF's, which may be extended to use a variety ofMEF's with selected genetic abnormalities; MCF-7 breast carcinoma cells;SY5Y neuroblastoma cells; and RKO colerectal carcinoma cells.

[0168] As a precursor for potential introduction of ATMdominant-negative cDNA's into tumors in vivo, any of the above ATMfragments that exhibit dominant-negative activity can be introduced intoa variety of tumor cells, e.g., with adenoviral vectors, AAV, retroviralvectors, or lentiviral vectors. Inhibition of ATM activity is assessedas described above. Such vectors can also be used to administer theATM-inhibiting gene to tumors in vivo and assess in vivoradiosensitization (see below).

ATM Function in Animal Models

[0169] Dominant-negative ATM cDNAs and ATM antagonists or agonists canbe used to initiate investigations in vivo relating to optimal use ofcandidates selected as a result of the screen for ATM kinase modulators,particularly inhibitors, or to evaluate ATM-mediated cellular processes.Animal models of the therapeutic modulation of ATM can also be preparedto evaluate ATM function.

[0170] Xenograft Models.

[0171] Xenografts of cell lines, particularly tumor cell lines, stablyor transiently expressing a dominant-negative ATM cDNA construct aregenerated. (Testing for loss of ATM-function is done in an in vitro cellassay, as described above.) Using mouse xenograft model systems,increased sensitivity of tumors expressing ATM-inhibitory activities isused to compare radiosensitivity of these tumors to tumors generatedfrom parental (unmodified) cell lines. Toxicity to normal tissues isexamined. The results of exposure to selected chemotherapeutic agents,particularly agents to which AT cells should exhibit increasedsensitivity (see above), is evaluated. Excellent mouse xenograft tumormodels have been developed (Zamboni et al., J. Natl. Cancer Inst.,90:505, 1998) for testing tumor cell sensitivity, and such model systemscan be employed for these studies.

[0172] “Brachytherapy” Models.

[0173] Brachytherapy is discussed in detail in the therapeutic sectionbelow.

[0174] Any number of different in vivo tumor model systems may be usedto test brachytherapeutic approaches. One preferred model system usesneuroblastoma cell lines. Antibodies directed against the GD2ganglioside are available and have been used both in vivo and in vitroto direct toxins or radioisotopes to tumor cells. Thus, pairedneuroblastoma cells with and without ATM dominant-negative vectors maybe generated, and their sensitivity to GD2-radioconjugates in thexenograft model system evaluated. Many other model systems and tumortypes can be similarly tested.

[0175] Animal model systems also allow characterization of directeddelivery of other cytotoxic agents in conjunction with ATM inhibition.In this case, ATM inhibition is effected in combination with anon-radioactive compound (which may be a highly desirable option forclinicians), for example, to explore conjugation of enedienes, likecalichearnicin or neocarzionstatin (NCS), to GD2 antibodies for testingin the neuroblastoma models. AT cells exhibit increased sensitivity toNCS and calichearnicin, and modified calicheamicins have been designedwith specific toxicity to tumor cells (Nicolaou et al., Science,256:1172-1178, 1992). In addition, calicheamicin θ¹ has beensuccessfully conjugated to GD2 antibodies and used to treatneuroblastoma in a mouse model system (Lode et al., Cancer Res.,58:2925-2928, 1998). Thus, such calicheamicin-conjugated anti-CD2 can beused in the xenograft models discussed above in conjunction with ATMinhibition by using the paired neuroblastoma models (with and withoutATM dominant-negative expression). Other antibodies and model tumorsystems can also be used, but the basic design and questions willbasically be the same. These experiments will facilitate use of a smallmolecule ATM inhibitor in conjunction with tumor-targeted cytotoxins.

Modulation of ATM Activity For Therapy

[0176] The present invention provides for modulating the activity of ATMkinase, either by inhibiting or increasing the level of ATM-mediatedphosphorylation, as indicated for treatment of a particular diseasestate. For example, for enhancing cellular radiosensitivity, inhibitionof ATM is desirable. Other pathological cellular processes, such aspremature aging, may be blocked or reduced by increasing ATM mediatedphosphorylation. Thus, a particular advantage of the present inventionlies in the ability to modulate ATM-regulated processes by modulatingATM-mediated phosphorylation of target proteins. Moreover, thespecificity of phosphorylation vis a vis other kinases, which has beendescribed above and in the Examples, infra, permits specificallytargeting cellular processes, but leaving other processes unaffected.

[0177] ATM activity can be inhibited by various means, including bydelivery of a dominant-negative ATM derivative (e.g., a kinase-deadmutant) to cells, by antisense nucleic acids (including ribozymes andtriple-helix-forming oligonucleotides; these are described in detailsupra), and by expression of anti-ATM intracellular antibodies, e.g.,single chain Fv antibodies (see generally Chen, Mol. Med. Today3:160-167, 1997; Spitz et al., Anticancer Res. 16:3415-3422, 1996;Indolfi et al., Nat. Med. 2:634-635, 1996; Kijima et al., Pharmacol.Ther. 68:247-267, 1995). In an alternative, small molecules discoveredby methods of the present invention and peptides identified herein canbe used to inhibit ATM activity.

[0178] ATM activity can be enhanced by increasing the level ofexpression of ATM in a cell, in vitro or in vivo. In a specificembodiment, the level of ATM activity is increased by transferring anexpression vector for ATM to the cell. In another embodiment, smallmolecules discovered by methods of the present invention can be used toinduce ATM activity.

[0179] Any of the vectors and delivery methods disclosed above can beused for modulation of ATM activity, e.g., in a therapeutic setting. Asdisclosed infra, the therapeutic methods of the invention are optimallyachieved by targeting the therapy to the affected cells. Means fortargeting delivery of various treatments, such as radiation orchemotherapy, are described below. However, in another embodiment, anATM inhibitor or an ATM stimulator can be targeted to cells, e.g., usingthe vectors described above in combination with well-known targetingtechniques, for expression of ATM modulators.

[0180] Furthermore, any of the therapies described herein can be testedand developed in animal models. Thus, the therapeutic aspects of theinvention also provide assays for ATM function.

Cancer

[0181] ATM is a particularly attractive target for inhibition in aclinical setting because the increased radiosensitivity of cells lackingATM function includes the low doses of radiation (1-2 Gy) typically usedclinically for tumor therapy. In addition, multiple doses or low-doserate (brachytherapy) might be expected to enhance the effects ofsensitization associated with ATM inhibition.

[0182] Radiosensitize Tumors to External Beam Irradiation.

[0183] Systemic application of this technology to all forms of cancercould be problematic because of increased toxicity to normal tissuesresulting from ATM inhibition. However, the radiosensitivity of both ATpatients and AT-knockout mice appears to be largely confined to the GItract, so the increased radiosensitization of normal tissues may notpresent a problem if the GI tract (mouth to rectum) is not in theradiation field. Thus, primary usage for tumors like brain tumors(glioblastoma multiforme in particular) and peripheral tumors such asbone tumors is envisioned. Moreover, with well-focused beams, one couldinhibit ATM in other tumors, like breast and lung carcinomas. ATMinhibition can also enhance sensitivity of metastatic lesions, e.g.,such as bony metastases in prostate cancer.

Brachytherapy by Local Delivery of Radioconjugates andChemotherapeutics.

[0184] Directing the cytotoxic exposure directly to the tumor itself isa commonly used approach to deliver a cytotoxic drug while minimizingthe cytotoxic exposure of normal tissues. However, one of the factorswhich limits the effectiveness of such an approach is incompleteinduction of tumor cell death because of limited dose delivery. Thus, itwould be highly desirable to concurrently use an ATM inhibitor toenhance the sensitivity of the tumor cells to the particular cytotoxicagent. Concurrent use of ATM inhibitor with the tumor-targeted deliveryof a radioisotope in an animal model is particularly indicated. Tumorspecific delivery is commonly achieved by conjugating a cytotoxic agentto an antibody that preferentially targets the tumor. Cytotoxic agentsinclude toxins (such as ricin) and radioisotopes. An ATM inhibitor isdesirable for targeted radiotherapy because radiation is long-term andlow dose, and ATM inhibition would be expected to effectively sensitizecells to this dose delivery mode. The targeting may be done with naturaltargeting (i.e., with radioactive iodine in the treatment of thyroidcarcinoma), physical targeting (i.e., administration of a radioisotopeto a particular body cavity), a tumor-specific antibody (e.g., anti-CD2in neuroblastoma or anti-Her2-neu in certain breast carcinomas), orother targeting protein (e.g., ferritin in hepatocellular carcinoma).

[0185] Using the same concepts discussed above for radioisotopes, localdelivery of certain chemotherapeutic agents could be used in combinationwith ATM inhibition. For example, AT cells exhibit enhanced sensitivityto enedienes such as calicheamicin, presumably because these drugsinduce DNA strand breaks. Thus, conjugation of calicheamicin to anantibody directed against GD2 (Lode et al., Cancer Res., 58:2925, 1998)could be used in combination with an ATM inhibitor to treatneuroblastoma.

[0186] This approach could similarly be used for any other tumor forwhich a specific antibody exists (e.g., anti-Her2neu in breast cancer).

Cardiovascular Disease

[0187] A major problem facing cardiologists following the use of balloonangioplasty to open up blocked coronary arteries is re-stenosis of thosearteries over the ensuing several months. One approach to inhibit suchrestenosis currently under investigation involves administration ofradioisotopes via catheter to the coronary artery after angioplasty totry to inhibit endothelial cell proliferation. This appears to work wellin porcine models. Concomitant administration of an ATM inhibitor withthe radioisotope would enhance the anti-proliferative effects.

[0188] Another approach is to use anti-proliferative drugs capable ofblocking endothelial cell proliferation. For example, a drug likecalicheamicin, which induces DNA strand breaks, would be effective,particularly in combination with an ATM inhibitor to enhance theeffectiveness of the drugs. This chemotherapeutic approach reducesdependence on radiation oncologists and physicists.

Revascularization

[0189] Furthermore, the “T” in AT stands for “telangiectasias”, which isproliferation of small blood vessels. Interestingly, in AT patients,these tend to occur on sun-exposed areas, like facial skin and sclerae.Clinically, AT patients appear to have a propensity to neoangiogenesisin mucosal areas exposed to toxic insults. For example, GI mucositis canlead to bleeding varicies months after the chemotherapy-inducedmucositis, and hemorrhagic cystitis appears months after treatment of ATpatients with Cytoxan. In both cases, this appears to represent a“hyperproliferation” of blood vessels in response to mucosal injury andreflects the same pathophysiology which results in telangiectasias insun-exposed areas in AT patients. Thus, it is envisioned that not onlywill an ATM inhibitor used concomitantly with a radioisotope orcytotoxic drug in a coronary artery after angioplasty inhibit restenosismore effectively, it might also result in helpful neoangiogenesis, e.g,restoration of blood vessels after treatment of an occlusion.

Obesity

[0190] It has been demonstrated that the ATM kinase phosphorylates aprotein called PHASI. The site in PHASI which gets phosphorylated hasbeen identified as serine 94. Interestingly, PHASI is involved intranslational regulation and is a critical part of signaling from theinsulin receptor. Highly phosphorylated PHASI is expressed at very highlevels in adipocytes, and is expressed at low levels or is nearly absentin other cell types. There are two closely related family members,PHASII and III, which are expressed in many cell types. Moreover, thoughthese three proteins are highly homologous, the serine 94 (target sitefor ATM) is present only in PHAS I. This ATM phosphorylation of PHASproteins in response to insulin may only occur in adipocytes.

[0191] It has been clinically reported that there is a mild form ofinsulin resistance in AT patients, especially those on steroids, whichis poorly understood mechanistically. The clinical observation has alsobeen made that AT patients are extraordinarily thin and have virtuallyno subcutaneous fat. The present discovery, that ATM phosphorylatesPHASI, suggests that lack of ATM function in AT patients preventsinsulin signaling to adequately stimulate growth of adipocytes.Therefore, inhibition of ATM may prevent insulin signaling in adipocytesand may thus be used to treat obesity. Other ATM target proteinsspecific for adipocytes could be reasonable targets to inhibit inreducing obesity or fat generation, and the present invention permitsidentification of such targets. Targeting ATM inhibition to adipocytesmay prevent adipocyte growth without resulting in systemic exposure.

Retroviral Infections, Including HIV

[0192] ATM is a target in the development of treatments for retroviralinfections, particularly human T cell leukemia virus (HTLV) infections.HTLV-I is linked to the generation of a certain form of T-Cell leukemia,and HTLV-III, also known as human immunodeficiency virus (HIV), is thecausative agent of acquired immune deficiency syndrome (AIDS). An ATMinhibitor may be used to block retroviral infection by leading to thedeath of cells into which an HTLV virus attempts to integrate.

[0193] DNA-PK is necessary for the repair of double-stranded DNA breaks.It has recently been reported that attempted retroviral integration incells that lack DNA-PK results in apoptosis (Daniel et al., Science,284:644-647, 1999). Cell death is due to unrepaired double-stranded DNAbreaks that arise during retroviral integration. Retroviral integrationis thus lethal to double-strand break repair deficient cells.

[0194] DNA-PK shares structural and functional properties with ATM,including the presence of a protein kinase domain in the carboxylterminal region of the protein. In addition, both enzymes appear to beinvolved in the repair of DNA double strand breaks. ATM deficient cellsdo not tolerate the integration of exogenous DNA and are sensitive toagents that cause DNA double strand breaks. Based on the presentdiscovery, retroviral integration is expected to be lethal toATM-deficient cells.

[0195] An inhibitor of ATM kinase, discovered by the screening procedureproposed in this application, may be used as a reagent for the treatmentof retroviral infections, particularly HTLV-I or HTLI-III (HIV)infections. HTLV-I and HTLI-III both integrate into the genome oflymphocytes. Treatment of lymphocytes with an ATM inhibitor is expectedto lead to death of the lymphocytes upon attempted retroviralintegration. The death of lymphocytes into which retroviruses attempt tointegrate thus has clinical utility for the treatment of either HTLV-Ior HTLV-III (HIV) infection, particularly during or after currentcombination therapy (with reverse transcriptase and proteaseinhibitors). ATM inhibition under these circumstances can “mop up” anyresidual, infectious virus to delay or prevent recurrence of high viraltiters and onset of AIDS.

[0196] One advantage of the use of an ATM inhibitor for the treatment ofHTLV infections is that the host cell, rather than the virus, istargeted for treatment. Targeting the host cells circumventsneutralization of many therapies resulting from the ability of the HIVvirus to rapidly mutate, leading to resistance to treatments that targetthe virus. HTLV viruses must integrate into a host cell for successfulinfection, and consequently would not be able to survive a drug thatleads to the death of any cell into which the virus attempts tointegrate.

[0197] An ATM kinase inhibitor may work in concert with currenttreatment regimes that target the HIV virus and is expected to reduce orprevent the ability of the virus to infect new lymphocytres. ATMinhibitors thus are clinically useful adjunct approaches to thetreatment of retroviral infections such as AIDS or HTLV-I inducedleukemias.

EXAMPLES

[0198] The present invention will be further understood by reference tothe following examples, which are provided as exemplary of the inventionand not by way of limitation.

Example 1 An in vitro Assay For ATM Kinase Function

[0199] ATM Constructs.

[0200] The full-length cDNA encoding NH₂-terminal, FLAG-tagged wild-typeATM (Ziv et al., Oncogene, 15:159-167, 1997) was excised from pFB-YZ3and subcloned into the XhoI site of pcDNA3 (Invitrogen) generatingpcDNA-FLAG-ATMwt. To generate catalytically inactive ATM, a cDNAfragment encoding the P13 kinase-related domain of ATM (Morgan et al.,Mol. Cell. Biol., 17:2020-2029, 1997) was mutated by overlap PCRsubstituting Asp 2870 with Ala and Asn 2875 with Lys. A cDNA fragmentencoding the kinase domain was excised from wild type ATM and replacedwith a Bpu1101 I-XhoI fragment containing the mutations described abovegenerating pcDNA-FLAG-ATMkd.

[0201] Transfection and Kinase Activity Assays.

[0202] 293T cells were transiently transfected with 10 μg of eitherpcDNA-FLAG-ATMwt, pcDNA-FLAG-ATMkd, pBJF-FRPwt or pBJF-FRPkd (Cliby etal., EMBO J., 17:159, 1998) using calcium phosphate and harvested twodays later. Cells were lysed through sonication in TGN buffer [50 mMTris (pH 7.5), 50 mM glycerophosphate, 150 mM NaCl, 10% glycerol, 1%Tween 20, 1 mM NaF, 1 mM NaVO₄, 1 mM PMSF, 2 μg/mg Pepstatin A, 5 μg/mlleupeptin, 10 μg/ml Aprotinin and 1 mM DTT] as described (Brunn et al.,Science, 277:99-101,1997). After centrifugation at 13,000× g, 2 mg ofextract were incubated with mouse IgG and protein A/G sepharose beads(Calbiochem). FLAG-tagged proteins were then immunoprecipitated withanti-FLAG M2 monoclonal antibody (Eastman Kodak Company) and protein A/Gsepharose beads. Immunoprecipitates were washed twice with TGN buffer,once with 100 mM Tris (pH 7.5) plus 0.5 M LiCl, and twice with kinasebuffer [10 mM Hepes (pH 7.5), 50 mM glycerophosphate, 50 mM NaCl, 10 mMMgCl₂, 10 mM MnCl₂, 5 mM ATP, and 1 mM DTT]. Kinase reactions wereinitiated by resuspending washed beads in 30 μl of kinase buffercontaining 10 μCi [γ-³²P]ATP and 1 μg GSTp53₁₋₁₀₁ and incubated for 30minutes at 30° C. Proteins were electrophoretically separated bySDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose,and analyzed on a Phosphorlmager. FLAG-tagged proteins and GSTp53₁₋₁₀₁were subjected to immunoblotting with either anti-FLAG M2 antibody ormonoclonal antibodies directed towards the NH2-terminus of p53 (Ab-2 andAb-6, Calbiochem) as described (Siliciano et al., Genes Dev., 11:3471,1997).

Example 2 Activation of ATM Kinase by Ionizing Radiation

[0203] The p53 tumor suppressor protein is activated and phosphorylatedon Ser¹⁵ in response to various DNA damaging agents. The gene productmutated in Ataxia telangiectasia, ATM, acts upstream of p53 in a signaltransduction pathway initiated by ionizing irradiation. The presentExample demonstrates that immunoprecipitated ATM had intrinsic proteinkinase activity and phosphorylated p53 on Ser¹⁵ in a Mn⁺²-dependentmanner. Ionizing radiation, but not ultraviolet radiation, rapidlyenhanced this p53-directed kinase activity of endogenous ATM. Theseobservations, along with the fact that phosphorylation of p53 on Ser¹⁵in response to ionizing radiation is reduced in AT cells, suggest thatATM is a protein kinase that phosphorylates p53 in vivo. Furthermore,the system used to evaluate phosphorylation of p53 is generallyapplicable in screening assays of other ATM kinase target proteins, andcould be used for screening for compounds that modulate ATM-mediatedphosphorylation. These data are published (Canman et al., Science281:1677-1679, 1998).

Materials and Methods

[0204] Phosphorylation of Ser¹⁵ of p53 by ATM and ATR/FRP1 in vitro.

[0205] 293T/17 cells were transfected with expression vectors encodingFLAG-tagged wild type (wt) or catalytically inactive (kd) ATM orATR/FRP1. After 48 hours, ATM or ATR was immunoprecipitated and used inthe in vitro kinase assay with [γ³²P]ATP and either wt, S6A, S9A, orS15A GSTp53₁₋₁₀₁ as substrates. Proteins from each reaction wereseparated by SDS polyacrylamide gel electrophoresis (7% gel),transferred to nitrocellulose, and analyzed either on a Phosphorlmageror by immunoblotting. Amounts of FLAG-tagged ATM or ATR in each kinasereaction were measured by immunoblotting with anti-FLAG M2 (top panel)and amount of [γ³²P]phosphate incorporated into ATM or ATR during thereaction (lower panel). Levels of phosphorylation in the in vitro kinaseassay with wt GSTp53₁₋₁₀₁ or various mutant GSTp53₁₋₁₀₁ proteins (S6A,S9A, or S15A) as substrates were determined by immunoblotting for p53.An upper immunoreactive band represents phosphorylated GSTp53 fusionprotein. The same exposures were used for ATM, ATR/FRP1 andcorresponding substrate proteins.

[0206] Fusion Proteins.

[0207] GSTp53₁₋₁₀₁ fusion protein was made by amplifying cDNA encodingthe first 101 amino acids of human p53 by PCR and subcloning thefragment into the EcoRI and BamHI sites of pGEX-2T (Pharmacia). Serines6, 9, or 15 were substituted with alanine using the QuikChangeSite-Directed Mutagenesis kit according to manufactures suggestions(Stratagene). GSTp531₁₋₁₀₁ wild type and mutant proteins wereindividually expressed in bacteria and purified onglutathione-conjugated agarose beads.

[0208] Posttranslational Modification of p53 on Ser¹⁵ in Response toIonizing Radiation.

[0209] Monoclonal antibodies against a chemically synthesized p53phosphoserine-15 peptide (amino acids 9-22) were used to immunoblotsynthetic peptides (1×, 50 μg) consisting of the first 24 amino acids ofp53 with (1-24^(S15-P)) or without (1-24) phosphoserine 15. Normal WT(2184) or AT (1526) lymphoblasts were untreated or treated with 5 Gy IR(IR) or 20 mM ALLN for 90 min. p53 was immunoprecipitated, subjected toSDS-PAGE (7.5% gel), transferred to nitrocellulose, and immunoblottedwith the monoclonal antibody to phosphoserine 15 p53 (upper panel).Blots were then stripped and immunoblotted with antibodies to p53 (lowerpanel).

[0210] Normal (2184) and AT (1526) EBV immortalized human lymphoblastswere irradiated with a ¹³⁷Cs source or treated with 20 mm ALLN (Sigma)for 90 min. Cells were then harvested, lysed, and p53 wasimmunoprecipitated as described (Siliciano et al., Genes Dev., 11:3471,1997). Immunoprecipitates were resolved by non-reducing SDS-PAGE,transferred to nitrocellulose and immunoblotted with monoclonal antibodyto phosphoserine 15. Blots were stripped and reprobed with p53-specificmonoclonal antibodies (Ab-2 and Ab-6).

[0211] Assay For Activation of Endogenous ATM Kinase by IonizingRadiation in vivo.

[0212] 2184 or 536 individual normal lymphoblasts or 1526 ATlymphoblasts (AT) were either untreated or treated with 5 Gy IR andharvested 20 or 60 min later. ATM was immunoprecipitated and assayedwith wild type GSTp53₁₋₁₀₁ protein as a substrate. Amounts of ATMpresent in each reaction were determined by immunoblotting with anti-ATM(Ab-3) and the amount of radiolabel incorporated into ATM during thekinase reaction was visualized with a PhosphorImager. Amounts of[γ³²P]phosphate incorporated into GSTp53₁₋₁₁₀ during each reaction wasvisualized with a Phosphorlmager (upper panel). Serine 15phosphorylation of GSTp53₁₋₁₀₁ was determined by immunoblotting withanti-phosphoserine 15 p53. 2184 and 536 lymphoblasts were treated withIR or 10 J/m² UV. Endogenous ATM was immunoprecipitated and used in anin vitro kinase assay with GSTp53₁₋₁₀₁ as substrate. The amount of³²P-labeled GSTp53₁₋₁₀₁ was quantitated with a Phosphorlmager andnormalized to that obtained with immunoprecipitates from unirradiatedcells.

[0213] Cells were either irradiated or treated with ultravioletirradiation as described (Canmann et al., Cancer Res., 54:5054, 1994).Endogenous ATM was immunoprecipitated from 3 mg of lysate withATM-specific rabbit polyclonal antibody (Ab-3, Calbiochem) and subjectedto the in vitro kinase assay. The beads and reaction mixtures wereseparated, resolved by SDS-PAGE and transferred onto Immobilon-P(Millipore) for ATM or nitrocellulose for GSTp53₁₋₁₀₁. Radiolabeledproteins were visualized and quantitated on a Phosphorlmager usingImageQuant software (Molecular Dynamics). Membranes were thenimmunoblotted with antibodies to ATM (Ab-3) or phosphoserine 15.

Results

[0214] We tested whether ATM might also phosphorylate p53 on Ser¹⁵ andwhether the activity of ATM towards p53 as a substrate is regulated byionizing irradiation. Furthermore, most naturally occurring ATM mutantproteins are unstable (Watters et al., Oncogene, 14:191, 1997). Becausea catalytically-inactive ATM mutant is a critical control for in vitrokinase assays, we constructed such a mutant that can be stablyexpressed. The putative kinase domain of ATM resides in theCOOH-terminus of the protein. In related proteins, three critical aminoacids within this domain are necessary for phosphotransferase activity(Savitsky et al., Science, 268:1749, 1995; Hunter, Cell, 83:1, 1995).Thus, a recombinant, FLAG-tagged, wild-type ATM was used as a source ofATM protein and a FLAG-tagged, mutant ATM expression construct wasgenerated in which two of the three critical amino acid residuesrequired for catalysis were mutated (D2870A and N2875K). Wild-type andmutant recombinant ATM proteins were individually expressed in 293Tcells and in vitro kinase activity was assessed. Equivalent amounts ofwild type (wt) and mutant (kd) ATM recombinant proteins wereimmunoprecipitated and incubated with [γ³²P] ATP and recombinantglutathione S-transferase-conjugated p53 protein containing the first101 amino acids of p53 (GSTp53₁₋₁₀₁). Although the proteins could bedetected with an anti-FLAG or anti-p53 antibody, only the wild typeenzyme phosphorylated GSTp53₁₋₁₀₁. ATM did not phosphorylate GST alone.

[0215] Endogenous p53 becomes phosphorylated on Ser¹⁵ and one otherserine residue within the first 24 amino acids of the protein inresponse to IR (Siliciano et al., Genes Dev., 11:3471, 1997). We testedwhether mutation of each of the four serine residues (S6, S9, S15, S20)within the first 24 amino acids of p53 altered the ability of ATM tophosphorylate the NH₂-terminus of p53. Recombinant ATM wasimmunoprecipitated and used to phosphorylate wt or mutant GSTp53₁₋₁₀₁.Wild-type recombinant ATM phosphorylated wt p53, S6A and S9A mutant p53but not S5A mutant p53 protein. Similar results were obtained withsynthetic peptides comprising the first 24 amino acids of p53 (data asabove). Therefore, ATM or a closely associated kinase phosphorylatesGSTp53₁₋₁₀₁ exclusively on Ser₁₅ in vitro. Wild-type ATM kinase alsoshowed autophosphorylation in this assay. Because mutation of Asp2870and Asn2875 within the kinase domain of ATM abolished bothphosphorylation of p53 and autophosphorylation of ATM, the kinaseactivity observed in these assays appears to be intrinsic to the ATMprotein. The DNA-PK also phosphorylates Ser¹⁵ (Lees-Miller et al., Mol.Cell. Biol., 12:5041, 1992), but unlike DNA-PK, ATM was dependent uponthe presence of Mn⁺² and did not require the addition of exogenous DNAfor activity (as above).

[0216] ATR/FRP-1, another PI-3-kinase related family member, may sharefunctional overlap with ATM in cell cycle checkpoint function (Keegan etal., Genes Dev., 10:2423, 1996; Cliby et al., EMBO J., 17:159, 1998).Conditional expression of catalytically-inactive ATR/FRP-1 abrogatesG2-M cell cycle arrest in response to IR. Furthermore, overexpression ofwild-type ATR/FRP-1 complements the defective IR-inducible S-phasecheckpoint in AT cells. Although ATM is required for rapidphosphorylation of Ser¹⁵ in response to IR in vivo, ATM appears not tobe required when cells are exposed to other genotoxic agents, such as UVirradiation. Thus, other cellular kinases must also phosphorylate p53 onSer¹⁵ in vivo. FLAG-tagged recombinant wt ATR/FRP-1 also showedautophosphorylation in vitro that was dependent upon the integrity ofthe catalytic domain. Like ATM, ATR/FRP-1 phosphorylated p53 on Ser¹⁵ ina Mn⁺²-dependent manner, though ATR/FRP-1 had at least 20-fold lessactivity than ATM towards GSTp53₁₋₁₀₁ when assayed under identicalexperimental conditions. Thus, p53 appears to be a better substrate forATM as compared to ATR/FRP-1.

[0217] To test whether endogenous p53 required ATM for phosphorylationon Ser¹⁵ in cells treated with IR in vivo, a monoclonal antibodyspecific for p53 phosphorylated at Ser¹⁵ was generated. The p53 proteinwas immunoprecipitated from normal and AT lymphoblasts either exposed to5 Gy IR or treated with the proteosome inhibitor,acetyl-Leu-Leu-norleucinal (ALLN), which causes stabilization of p53protein. Immunoblot analysis with this antibody demonstrated that p53became phosphorylated only in normal lymphoblasts exposed to IR.Phosphoserine 15 was undetectable in normal cells treated with ALLN,although they accumulated equivalent amounts of total p53 protein tothose in irradiated cells. Phosphoserine 15 p53 was also undetectable inthe 1526 AT line. Thus, examination of radiation responses in ATM-mutantcells further supports this link between ATM and irradiation-inducedphosphorylation of p53.

[0218] Activation of endogenous ATM was examined in two different normallymphoblast cell lines exposed to 0 or 5 Gy IR. ATM immunoprecipitateswere used to phosphorylate GSTp53₁₋₁₀₁ in vitro. Within 20 min afterexposure to IR, ATM protein kinase activity toward GSTp53₁₋₁₀₁ wasincreased approximately 2-fold. This appeared to be an increase in thespecific activity of ATM because the amount of ATM protein did notchange in response to IR. Kinase activity towards p53 substrate wasminimal in immunoprecipitates from an AT lymphoblast line. TheIR-induced activity associated with ATM was directed to Ser¹⁵ becausethe immunoprecipitated endogenous ATM from irradiated cells increasedphosphorylation of Ser¹⁵ in in vitro kinase assays. Therefore, ATMkinase appears to be activated in response to IR and phosphorylates p53on Ser¹⁵.

[0219] Cells derived from AT patients are not hypersensitive to UVirradiation (Lavin and Shiloh, supra, 1997; McKinnon, Hum. Genet.,75:197, 1987). Furthermore, such cells respond normally to UV withincreased synthesis of p53, phosphorylation of p53 on Ser¹⁵, andactivation of the stress-activated SAP kinase (JNK) pathway (Canman, etal., Cancer Res., 54:5054, 1994; Kahanna and Lavin, Oncogene, 8:3307,1993; Siliciano et al., supra, 1997; Shafman et al., Cancer Res.,55:3242, 1995). The kinase activity of ATM was not increased in cellsexposed to UV irradiation. Slight activation of ATM kinase was detectedat more than 60 min after exposure, which may be due to signalsgenerated by DNA strand breaks associated with DNA repair (Nelson etal., Mol. Cell. Biol., 14:1815, 1994). These results confirm that ATMplays little role in the cellular UV response and suggests that anotherkinase other than ATM phosphorylates p53 on Ser¹⁵ in response to UVirradiation.

[0220] Previous genetic and biochemical evidence implicated the ATM geneproduct in regulating the phosphorylation and induction of p53 in cellsexposed to ionizing radiation (Kastan et al., Cell, 71:587, 1992;Siliciano et al., supra, 1997; Xu et al., Genes Dev., 10:2401, 1996;Barlow et al., Nature Genetics, 17:453, 1997). Our results indicate thatATM is a protein kinase whose activity is increased by ionizingirradiation and whose in vivo target may be Ser¹⁵ of p53. Thisconclusion is consistent with the finding that ATM and p53 proteinsdirectly interact with each other (Watters et al., Oncogene, 14:1911,1997). The functional ramifications of radiation-induced Ser¹⁵phosphorylation remains to be clearly elucidated. However,phosphorylation of p53 on Ser¹⁵ reduces binding of the mdm2 oncogeneproduct to p53 in vitro (Shieh et al., Cell, 91:325, 1997) and bindingof mdm2 to p53 promotes rapid degradation of p53 by targeting it forproteolytic degradation, thereby potentially controlling p53 proteinlevels (Haupt et al., Nature, 387:296, 1997; Kubbutat et al., ibid, p.299). Because many of the clinical manifestations exhibited by ATpatients can not be attributed to abnormal regulation of p53 alone,other important targets of the ATM kinase are identified herein.

Example 3 Examination of ATM Kinase Family Substrate Specificities

[0221] From the ATM kinase substrate site identified for p53, homologouscandidate sites in other proteins were identified and tested, firstusing the GST-peptide approach, and then with the full length or nearlyfull length target. This approach identified a number of ATM targetproteins.

[0222] Based on the fact that p95/nibrin is mutated in an A-T likesyndrome, the Nijmegen breakage syndrome, this Example also reports itsevaluation as a valid in vitro target. Ionizing irradiation of cellscauses ATM-dependent phosphorylation of serine 343 of p95 protein,identical to the in vitro target site for ATM. These observationsconfirm that ATM participates in signaling to a protein in sensing ormodulating repair of DNA damage.

Materials and Methods

[0223] Antibodies.

[0224] The anti-Flag M2 and anti-c-Myc monoclonal antibodies wereobtained from Sigma Co. and Roche, respectively. The p95 rabbitpolyclonal antibody was described previously (Carney et al., Cell,93:477-86, 1998). The a-p95-phosphoserine 343 polyclonal antibody wasgenerated by immunizing mice with a KLH-conjugated phosphopeptide(TPGPSL(PO3)SQGVSVDE) (SEQ ID NO:10). The a-p95-phosphoserine 343antiserum was purified by affinity chromatography usingphosphopeptide-conjugated Sepharose and antibody components directedagainst non-phosphospecific epitopes were removed on a Sepharose columnconjugated with an unphosphorylated peptide (TPGPSLSQGVSVDE) (SEQ IDNO:10). When used in Western blots, the antibody was pre-incubated withthe unphosphorylated peptide to block any residual reactivity withunphosphorylated p95. The ATM monoclonal antibodies (D16.11 forimmunoprecipitation, and D16.35 for immunoblotting) are described above.

[0225] Plasmids and GST-Fusion Protein Production.

[0226] For GST fusion peptide expression vectors, complementaryoligonucleotides encoding desired peptides (14 amino acids) were clonedinto the BamHI/SmaI site of pGEX-2T (Pharmacia). The constructs wereconfirmed by restriction enzyme digests and DNA sequencing. To constructthe larger GST-p95 (327-391) protein fragment, p95 DNA encoding aminoacids 327-391 was amplified by PCR with Pfu polymerase using thefollowing primers: 5′ sense, 5′ sense5′-TCCCCAGGAATTCCCGGCCATCCCAGTACAGGATTA-3′ (SEQ ID NO:45), 3′ antisense,5′-TGCGGCCGCTCGAGTTTTTTGTTCCATTTTGGAGAC-3′ (SEQ ID NO:47).

[0227] The amplified PCR product was digested with EcoRI/XhoI and wascloned into pGEX-4T-2 (Pharmacia). GST-p95 (327-391) S343A was generatedusing the QuikChange Site-Directed Mutagenesis kit (Stratagene). Toconstruct the Myc-tagged p95 expression vector, the entire coding regionof p95 was PCR-amplified with a pair of oligonucleotides:5′-GAATCCCTCGAGCCTACCGCCATGTGGAAACTGCTGCCCGCCGCG-3′ (SEQ ID NO:47) and5′-GTCGACGAGCGGCCGCCACCTCAGGGATCTTCTCCTTTTTAAATAAGG-3′ (SEQ ID NO:48)

[0228] NO:48). The PCR product was digested with XhoI/NotI and clonedinto a pSG5 vector (Neupogen) that had a c-Myc tag inserted. TheGST-peptides or fusion proteins were expressed in BL21(DE)3 and purifiedon glutathione-Sepharose beads.

[0229] In vitro Kinase Assays and Immunoprecipitation.

[0230] In vitro kinase assays for wild-type and catalytically inactiveATM and ATR were performed as described above. Briefly, cell extractswere prepared from 293T cells which had been transfected with 10 mg ofeither ATM or ATR by resuspending cells in modified TGN buffer [50 mMTris (pH 7.5), 150 mM NaCl, 1% Tween 20, 0.3% NP-40, 1 mM NaF, 1 mMNa₃VO₄, 1 mM PMSF, and lx protease inhibitor mixture from Roche].Cleared supernatants were immunoprecipitated with anti-Flag M2 antibodyand protein A/G agarose, the beads were washed with TGN buffer followedby TGN buffer plus 0.5 M LiCl, and two washes with kinase buffer [20 mMHEPES (pH 7.5), 50 mM NaCl, 10 mM MgCl₂, 1 mM dithiothreitol (DTT) and10 mM MnCl₂]. Finally, the immunoprecipitant was resuspended in 50 ml ofkinase buffer containing 10 μCi of [γ-³²P] ATP and 1 mg of GST-fusionsubstrate (GST-p53:1-101), in varying concentration of Mn²⁺. The kinasereaction was conducted at 30° C. for 20 min and stopped by the additionof SDS-PAGE loading buffer. Proteins were separated onSDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred tonitrocellulose. Radiolabeled proteins were visualized and quantitated ona Phosphorlmager (Molecular Dynamics). Use of equivalent amounts offlag-tagged ATM or ATR in the different lanes was confirmed byimmunoblotting with anti-flag antibody. In some reactions, eithersupercoiled DNA (pBluescript II KS, Stratagene) or linearized DNA(pBluescript II KS cut with EcoRI) was added (0, 0.2, 0.5, 1, and 2 mg).For endogenous ATM kinase reactions, endogenous ATM wasimmunoprecipitated from GM0536 lymphhoblasts with ATM monoclonalantibody (D16.11) in M buffer (PBS, 10% glycerol, 0.2% Tween 20, 0.3%NP-40, 1 mM NaF, 1 mM Na₃VO₄, 1 mM PMSF, 1× protease inhibitor mixture].After washing with M buffer and kinase buffer, in vitro kinase reactionswere carried out according to procedures described above. The DNA-PK invitro kinase reaction was performed as previously described (Gottlieband Jackson, 1993), using 20 ng each of DNA-PKcs or DNA-PK (20 ngDNA-PKcs premixed with 20 ng Ku 70/80).

[0231] For immunopreciptation of p95 and ATM, 106 cells were harvestedin lysis buffer [10 mM Tris HCl (pH 7.5), 200 mM NaCl, 5 mM EDTA, 0.5%NP-40]. After centrifugation, supernatants were incubated followingstandard protocols with either anti-p95 antibody or anti-ATM antibody.After extensive washing with a washing buffer [10 mM Tris HCl (pH 7.5),100 mM NaCl, 5 mM EDTA, 0.5% NP-40], immunoprecipitants were analyzed byimmunoblot. For lambda phosphatase treatment, immunoprecipitated p95 waswashed with a phosphatase buffer and incubated with 400 units of lambdaphosphatase at 30 C. for 30 min.

[0232] Phosphorylation of Large GST-p95 Protein Fragment by ATM invitro.

[0233] Either GST-p95 (327-391) or GST-p95 (327-391) with serine at 343substituted by alanine (S343A) was used as a substrate for ATM in vitrokinase reactions. Either transfected, flag-tagged wild-type (wt) orkinase-dead (kd) ATM or endogenous ATM from normal (WT; GM0536) or ATlymphoblasts (GM1526) was used as the source of ATM kinase. Flag-taggedATM or endogenous ATM in each kinase reaction was blotted with anti-Flagor anti-ATM. The amount of phosphorylation of ATM and substrate wasanalyzed on a Phosphorlmager.

[0234] In vivo Phosphorylation of p95.

[0235] Myc-tagged p95 (3 μg) was transiently co-transfected into 293Tcells with wtATM, kdATM, or pcDNA3 (7 μg) using calcium phosphate. After2 days, cells were harvested and Myc-p95 was immunoprecipitated,subjected to SDS-PAGE (7.5%), transferred to nitrocelluose, andimmunoblotted with α-cMyc antibody or the α-p95-phosphoserine 343polyclonal mouse antibody. For metabolic labeling, the cells were washedwith phosphate free RPMI plus 10% dialyzed fetal bovine serum andincubated for 30 min before irradiation. The cells were irradiated at 5Gy and incubated with [³²P] orthophosphate (1 μCi/ml) for 30 min at 37°C. The labeled cells were lysed at 4° C. in TGN buffer and Myc-p95 andFlag-ATM were immunoprecipitated. After electrophoresis in 7.5%SDS-PAGE, radiolabeled p95 and ATM were visualized and quantified on aPhosphorlmager.

Results

[0236] Cofactor Requirements For ATM, ATR and DNA-PK.

[0237] In order to elucidate potentially distinctive biochemicalproperties of the ATM, ATR, and DNA-PK kinases, we examined the cofactorrequirements for their optimal activities. It had been previouslyreported that ATM and ATR required exogenous Mn²⁺ for optimal in vitrokinase activity (Banin et al., Science, 281:1674, 1998; Canman et al.,Science, 281:1677, 1998). In contrast, DNA-PK has been reported torequire Mg²⁺ as well as the presence of DNA-ends and the DNA-bindingproteins Ku70 and Ku80 for optimal activity (Gottlieb and Jackson, Cell,72:131, 1993). However, since ATM specific activity increases in cellsfollowing exposure to ionizing radiation (Canman et al., supra, 1998) orintroduction of double strand DNA breaks (Banin et al., supra, 1998), itwas possible that ATM or ATR kinase activities are enhanced by thepresence of DNA ends and that this dependence was being missed in our invitro assays. For example, it was conceivable that the use of Mn²⁺ inthese assays decreased the dependence of ATM and ATR on DNA ends.Conversely, it was conceivable that the requirement of the use of DNAends and/or Ku proteins in DNA-PK activity assays abrogated a need forMn²⁺ addition in these in vitro assays.

[0238] In order to test these possible scenarios, in vitro kinase assayswith all three enzymes were performed using epitope-tagged ATM and ATRimmunoprecipitated from transfected cells and biochemically purifiedDNA-PK as the kinase sources and GST-p53 (1-101) recombinant protein asa substrate. Kinase-inactive forms of ATM and ATR were used as controlsin the assay to ensure that these activities were intrinsic. ATM and ATRkinases were dependent on the addition of exogenous Mn²⁺, while DNA-PKactivity was not altered by the addition of Mn²⁺. The addition ofsupercoiled or linearized DNA to these reactions did not alter theactivity of ATM or ATR, but consistent with previous observations(Gottlieb and Jackson, 1993), the dsDNA ends provided by linearized DNAsignificantly enhanced the activity of DNA-PK. The addition of either ofthese exogenous DNA sources did not relieve the dependence of ATM andATR on exogenous Mn²⁺.

[0239] It remained theoretically possible that these overexpressed ATMand ATR proteins had different cofactor requirements than would be seenfor the normal endogenous proteins because of changes in molar ratios ofATM/ATR and cofactors. In addition, it was possible thatimmunoprecipitated ATM/ATR could be already contaminated with DNA ormight have lost cofactors such as Ku proteins duringimmunoprecipitation. Therefore we re-examined both the Mn²⁺ dependenceand DNA-end dependence using endogenous ATM. In order to circumvent theconcerns about potential loss of cofactors or prior DNA contamination,we also immunoprecipitated the endogenous ATM under very mild conditionswithout stringent washing (0.5M LiCl₂) and assessed the kinase activityeither in the presence of added linearized DNA or with the addition ofethidium bromide, a DNA intercalator which is known to interfere withprotein-DNA interaction (Schroter et al., EMBO. J., 4:3867, 1985; Laiand Herr, Proc. Natl. Acad. Sci. USA, 89:6958, 1992). The activity ofendogenous ATM was still dependent on the addition of exogenous Mn²⁺ andneither the exogenous addition of DNA ends nor the presence of ethidiumbromide to inactivate potential contamination from endogenous DNA hadany effect on its activity.

[0240] DNA-PK activity is enhanced by the presence of DNA ends viaKu-dependent association with the DNA and Mn²⁺ is not required for itsactivity in in vitro kinase assay (Gottlieb and Jackson, supra, 1993).To further clarify potential distinctions between ATM/ATR and DNA-PK andto rule out the possibility that Mn²⁺ might replace the requirement ofDNA-PK activity for DNA ends or for the Ku70/80 cofactors, the in vitroactivities of purified DNA-PKcs or DNA-PK holoenzyme containing Kucomponents were re-examined. Though DNA-PKcs alone has a basal level ofactivity toward GST-p53 (1-101), this activity was not enhanced by theaddition of either Mn²⁺ or DNA-ends. In contrast, the activity of theDNA-PK holoenzyme was remarkably enhanced by DNA-ends (approximately 10times of basal level) and the addition of Mn²⁺ had no effect on theDNA-PK activity either in the presence or absence of exogenous DNA.Thus, ATM and ATR kinases are distinguishable from DNA-PK in their lackof dependence on DNA-ends and their requirement for Mn²⁺ for optimalactivity.

[0241] Consensus Sequence Elucidation via GST-p53 Peptide MutagenesisAnalysis.

[0242] Studies on other kinases have suggested that the nature andsequence of the amino acids surrounding the target phosphorylation sitecan play a critical role in modulating recognition of a substrate by akinase (Pinna and Ruzzene, Biochim. Biophys. ACTA, 1314:191, 1996). Wewished to clarify amino acids that affect the ability of ATM tophosphorylate a substrate and to use this information to try to identifyother physiologic substrates. Short peptide sequences containing targetamino acids have commonly been used as in vitro substrates for kinases.As described above, linkage of a short peptide sequence to a GST-tagprovides a better in vitro substrate for ATM than a short peptide alone(presumably by increasing the size and secondary structure of the targetpeptide sequence). This approach also provided an easy way to generateand purify the peptide of interest. Thus, beginning with the known invivo and in vitro target, serine 15 in p53, we attempted to determinewhich amino acids surrounding Ser15 in p53 were important modulators ofphosphorylation by ATM. Wild type and mutated GST-conjugated p53peptides containing 14 amino acids surrounding Ser15 were generated andused as in vitro kinase substrates for ATM, ATR and DNA-PK. Changes insome of the amino acids surrounding serine 15 had a dramatic effect onthe ability of these kinases to phosphorylate the GST-p53 peptides,while others had little effect (FIGS. 1A-C). For example, peptides inwhich glutamine at position 16 was replaced with alanine, glycine, orarginine were very poor substrates for all three of these kinases. Thus,as has previously been reported for DNA-PK (Lees-Miller et al., Mol.Cell Biol., 12:5041, 1992), this result suggests that a glutamineadjacent to the target serine is also critical for substrate recognitionby ATM and ATR. When the target serine at position 15 was mutated intothreonine, the amount of phosphorylation was reduced to 13%, 53%, and36% compared to the wild-type sequence for ATM, ATR, and DNA-PK,respectively. Thus, all three kinases appear to have a preference forphosphorylating serine over threonine. All three kinases poorlyphosphorylated peptides with the substitutions 12D, 12R, 12K, 13K, 14K,and 14R, while the 12Q, 12A, 13A, 14A, 14Q, and 14D peptides wereefficiently phosphorylated. These results indicate that hydrophobicamino acids at positions N-3 and N-1 and negatively charged amino acidsat N−1 are positive determinants for substrate recognition by thesekinases. In contrast, positively charged residues (11K, 12K, 12R, 13K,14K, 14R, 17K, and 19K) around the SQ appear to have a significantnegative influence on substrate phosphorylation.

[0243] Though many of the amino acid substitutions had similar effectson all three of these kinases, some of the target manipulations resultedin differential effects. Peptides with 10Q, 17K, 17L substitutionsremained relatively good substrates for ATM and ATR, but were very poorsubstrates for DNA-PK (FIG. 1). These observations suggest that thepositions N−5 and N+2 are more important for substrate recognition byDNA-PK than for ATM and ATR. Most of the amino acid substitutions hadsimilar effects on ATM and ATR, though the substitutions 12A, 13A, 14Q,and 14D appeared to result in differential quantitative effects on invitro ATM and ATR activity (FIG. 1). One exception to this was thevirtual abrogation of ATM activity by valine substitution for leucine atN−1 with no obvious affect on ATR activity (data not shown). Thus, ATM,but not ATR, may discriminate between valine and other hydrophobic aminoacids at the position N−1. Further validation of this putative generalconsensus target sequence for ATM came from examination of a previouslyreported in vitro substrate for ATM, PhasI (Banin et al., supra, 1998).An amino acid sequence around Ser 94 in PhasI contains hydrophobic aminoacids at N−3 and N−1 as well as an SQ motif. As predicted, a GST-Ser94PhasI peptide proved to be an excellent in vitro substrate for ATM (FIG.2).

[0244] Identification of New Putative Substrates of the ATM Family.

[0245] Using the preliminary consensus sequence generated from the p53Ser15 peptide mutational analysis and the Ser94 sequence of PhasI, wesearched a protein database for potential ATM substrates. Since theoccurrence of this motif is not uncommon, many potential targets wereidentified. We initially evaluated the ability of ATM, ATR and DNA-PK tophosphorylate the peptide sequences of 36 proteins (Table 4), many ofwhich were chosen because of potential physiological relevance to the ATphenotype. Only a fraction of the peptides tested were highlyphosphorylated in vitro (FIG. 2). These included Rad17, p95, Brca1,PhasI, WRN, ser440 of ATM (ATM440, a potential autophosphorylationsite), and a recently cloned sequence localized to an LOH site onchromosome 3p21.3 (which we call PTS for “putative tumor suppressor”).Since all of the tested peptides contained sequences which fit thegeneral consensus derived from the mutational analysis above, otheramino acid sequence determinants must also be involved in determiningthe substrate specificity for the ATM kinase and continuedcharacterization of these substrates should allow further refinement ofthe recognition sequence.

[0246] Table 4—Amino Acid Sequences and Relative Phosphorylation by ATMKinase Family Members of the GST-Peptides

[0247] The sequences and accession numbers of the peptides studied arelisted and the amounts of substrate phosphorylation relative to thep53ser15 site by each kinase is shown. nd=not determined. RelativeAccession Protein Name Test Peptide SEQ ID Phosphorylation (%) Number(Amino acid sequences) NO. Serine No. ATM ATR DNA-PK p04637 p53SVEPPLSQETFSDL 7 S15   100 100 100 p53 VLSPLPSQAMDDLM 8 S37   9 31 132NP_004086 4EBP1 (Phas I) EPPMEASQSHLRNS 9 S94   78 66 10 NP_002476 p95TPGPSLSQGVSVDE 10 S343  19 31 45 p49959 MRE11 (SQ1) QQLFYISQPGSSVV 11S264  20 48 10 MRE11 (SQ2) FSVLRFSQKFVDRV 12 S386  <1 17 <1 MRE11 (SQb3)RARALRSQSEESAS 13 S531  <1 13 <1 MRE11 (SQ4) SASRGGSQRGRAFK 14 S590  <1nd nd MRE11 (SQ5) SSSKIMSQSQVSKG 15 S648  <1 nd nd NP_001265 CHK1 (SQ1)NVKYSSSQPEPRTG 16 S317  14 58 2 CHK1 (SQ2) VQGISFSQPTCPDH 17 S345  11 7320 AAC36334 RAD17 (SQ1) TWSLPLSQDSASEL 18 S646  81 733 97 RAD17 (SQ2)ASELPASQPQPFSA 19 S656  91 1065 25 AF091214 WRN (SQ2) TIGMHLSQAVKAGC 20S1292 10 33 5 WRN(SQ1) EKAYSSSQPVISAQ 21 S1141 25 49 14 U76308 ATRTVEPIISQLVTVLL 22 S1333 <1 10 <1 X16416 c-ABL YPGIDLSQVYELLE 23 S446  <18 <1 NP_000042 ATM PLLMILSQLLPQQR 24 S440  33 55 7 ATM YKVVPLSQRSGVLE 25S2761 3 22 <1 Z46973 PI-3K DLLMYLSQLVQALK 26 S397  <1 150 <1 U64105p115-RhoGEF RLRPLLSQLGGNSV 27 S899  <1 15 <1 L13939 β-ADAPTINCRAPEVSQHVYQAY 28 S935  5 22 2 U43746 BRCA2 KVSPYLSQFQQDKQ 29 S2156 <1132 <1 M81735 DNA POL-δ LPCLEISQSVTGFG 30 S717  <1 17 <1 U87269 p120E4FAPEPPVSQELPCSR 31 S355  11 30 2 U14680 BRCA1 (SQ1) SASLFSSQCSELED 32S1298 2 38 6 BRCA1 (SQ2) DCSGLSSQSDILTT 33 S1387 42 35 36 BRCA1 (SQ3)SSEYPISQNPEGLS 34 S1466 8 33 12 L07590 PP2A (protein LLHIPVSQFKDADL 35S61   <1 34 <1 phosphatase 2A) D79987 CUT1 GASPVLSQGVDPRS 36 S1615 9 205 AF040703 PTS (123F2) WETPDLSQAEIEQK 37 S61   111 40 33 X63071 DNA 5BQPEPPVSQSEISEP 38 S72   3 145 15 X83441 DNA LIG-IV DLKLGVSQQTIFSV 39S132  5 36 48 U24186 RPA34kD (SQ1) FPAPAPSQAEKKSR 40 S33   <1 5 <1RPA34kD (SQ2) IVPCTISQLLSATL 41 S52   <1 31 <1 RPA34kD (SQ3)TGNVEISQVTIVGI 42 S72   <1 8 <1 U81504 β3A Adaptin ELKPVLSQG 43 S1092 622 44 U72066 CtIP DPGADLSQYKMDVT 44 S664  12 12 2

[0248] We also tested the ability of DNA-PK and ATR to phosphorylatethese GST-peptides and several distinctions between substratesrecognized by these three kinases were apparent in the assays (FIG. 2).In general, ATM and ATR tended to recognize the same substrates, thoughquantitative differences were apparent in most cases. For example, eventhough ATR appears to be a much weaker kinase than ATM for mostsubstrates tested (about 10-20 fold lower activity), ATR exhibitedgreater activity than ATM for two sites in Rad17 and also demonstratedgood activity against sites from Brca2 and DNA-5B. The latter twopeptides were very poor substrates for both ATM and DNA-PK and thesecond SQ in Rad17 was not a good substrate for DNA-PK (FIG. 2).Conversely, peptides containing sequences from ligase IV and the regionsurrounding Ser37 of p53 were highly phosphorylated by DNA-PK, but werenot good substrates for ATM. From these approaches, we have developed apreliminary consensus target sequence which is recognized by ATM and wehave identified a group of proteins containing good in vitro targetsites for ATM which warrant further investigation as potential valid invivo targets of ATM (Detailed Description, supra).

[0249] Structural factors present in a full-length protein, but absentin the GST-peptides we tested, could also influence the ability of ATMto phosphorylate a target protein. Thus, the next logical step inelucidating valid targets of the kinase was to test the ability of ATMto phosphorylate much larger peptides or full-length proteins from theputative target list. GST-linked peptides containing over 100 aminoacids from the putative Rad17 (561-670) and Brca1SQ2 (1341-1440) siteswere excellent in vitro substrates for ATM and ATR. In contrast, GST-WRN(1099-1198) and purified recombinant full-length WRN protein were notphosphorylated. A similar approach was used to evaluate the sequence inp95 (nibrin). A large GST-peptide containing serine 343 of p95 was anexcellent in vitro substrate for ATM. Mutation of serine 343 to alaninein the target GST-p95 largely abrogated the ability of ATM tophosphorylate the protein, thus confirming this as an in vitro targetsite in the protein. These observations led to further investigation ofp95, Rad17 and Brca1 (Example 4), but not WRN, as potential physiologictargets of ATM and ATR. The results with p95 and ATM are describedbelow.

[0250] p95 Forms a Complex with ATM and p95 Phosphorylation After IR isNot Observed in AT Cells.

[0251] Because of the overlap of the physiologic abnormalities in AT andNBS (Nijmegen breakage syndrome), we were particularly interested indetermining whether the p95 protein (nibrin), which is mutated in NBS,is a true physiologic target of ATM. Potential interactions betweenendogenous ATM and p95 proteins were initially evaluated byco-immunoprecipitation experiments using untreated and irradiated (10Gy, 1 hr) K562 cells, with detection of p95 and ATM by Western blotting.Anti-p95 antibodies brought down detectable amounts of ATM protein fromcellular lysates, particularly after irradiation with no ATM detectedusing pre-immune serum (or no antibody) to precipitate. However, thereverse experiment using anti-ATM antibodies for the immunoprecipitationdid not reveal detectable amounts of p95 immunoprecipitating with ATM.Similarly, we were unable to demonstrate co-immunoprecipitation of ATMand p95 after transient co-transfection. These results support theexistence of physical interaction between ATM and p95 though it islikely to be weak and transient. However, it is not uncommon forkinase-substrate interactions to be relatively weak and transient. Thus,we remained interested in further examining the possibility that p95 isa physiologic substrate for ATM.

[0252] We observed a slower migration of p95 protein on SDS-PAGE afterexposing two different mammalian cells to IR (FIG. 3). Since phosphatasetreatment of the immunoprecipitated p95 eliminated this slowed migration(FIG. 3), it appeared that this altered migration represented anIR-induced phosphorylation of p95 protein. This concept was supported bythe observation that metabolic labeling of 293T cells with ³²p afterco-transfection of epitope-tagged ATM (Ftg) and p95 (Myc) revealed adoubling of ³²P incorporation into p95 after IR (5 Gy). In order tobegin to evaluate the potential role of ATM in this IR-inducedphosphorylation of p95, the IR-induced band shift was evaluated in ATcells. The altered p95 migration after IR failed to occur in AT cells(FIG. 3), thus implicating ATM in this phosphorylation event. However,demonstration of a direct phosphorylation of p95 by ATM after IR wouldrequire a more definitive approach.

[0253] The Phosphorylation of p95 on Ser343 is Dependent on ATM and isDefective in AT Cells in Response to Ionizing Radiation.

[0254] In order to investigate whether ATM kinase phosphorylates p95 inresponse to IR and whether the site in p95 which gets phosphorylated byIR is the in vitro ATM target site in p95 that we had previouslyidentified, we generated a polyclonal antibody which specificallyrecognizes p95 when it is phosphorylated at ser343 (as detected byblotting phosphorylated but not unphosphorylated 337-350 peptidecontaining phosphoserine or serine at position 343). The specificity ofthe antibody was illustrated by its ability to recognize the p95 peptidein its phosphorylated state, but not its unphosphorylated state, whenincubated with blocking peptide. Using this antibody, we were able toask whether IR induces phosphorylation of ser343 in p95 in vivo andwhether this phosphorylation event is dependent on ATM. Either emptyvector or wild-type or kinase-inactive (kd) forms of ATM wereco-transfected into 293T cells along with myc-tagged p95 andradiation-induced phosphorylation of ser343 in p95 was examined.Immunoblots of immunoprecipitated p95 using the α-p95-phosphoserine-343antibody revealed very low levels of p95ser343 phosphorylation inunirradiated cells and a dramatic increase in phosphorylation after IR(FIG. 4). Thus, serine 343 is at least one site in p95 which isphosphorylated in response to IR. Interestingly, when thekinase-inactive form of ATM was co-transfected with p95, itsignificantly inhibited this IR-induced phosphorylation of p95ser343(FIG. 4). This suggests that similar to ATR (Cliby et al., EMBO. J.,17:159, 1998; Wright et al., Proc. Natl. Acad. Sci. USA, 95:7445, 1998;Tibbetts et al., Genes Dev., 13:152, 1999), overexpression ofkinase-inactive ATM is functioning as a dominant-negative protein byinhibiting irradiation-induced p95 S343 phosphorylation by theendogenous ATM protein. (It is noted that overexpression of thiskinase-inactive form of ATM exhibits dominant-negative activity in manyother assays of ATM function as well.

[0255] Thus, IR treatment of cells results in phosphorylation of theconsensus ATM in vitro target site in p95 that we had previouslyidentified and this IR-induced phosphorylation event is blocked by useof a dominant-negative form of ATM. As a final step in demonstrating arequirement for ATM kinase in the IR-induced p95ser343 phosphorylation,we examined the ser343 phosphorylation of endogenous p95 in response toDNA damage in normal and AT cells. Endogenous p95 was immunoprecipitatedfrom unirradiated or irradiated normal (GM536) and AT lymphoblasts(GM1526) and subsequently examined by Western blotting with theanti-p95ser343 antibody. Phosphorylation of Ser343 of p95 was easilydetected in irradiated cells containing wild-type ATM, but no reactivitywith the phosphospecific antibody was detected in either unirradiatedcells or AT cells with or without irradiation (FIG. 4B). Equivalentamounts of total immunoprecipitated p95 protein were present under allconditions. These results demonstrated that p95 protein isphosphorylated on ser343 in an ATM-dependent manner in response toionizing radiation.

[0256] Experiments have confirmed the physiological relevance of ATMphosphorylation of p95. Overexpression of cDNA for a mutant form of p95at the ATM phosphorylation site (Ser 343→Ala) altered the radiationresponse of the cells.

Discussion

[0257] Identification of physiologic substrates for cellular kinases isa daunting, but critically important, aspect of understanding biologicalprocesses. It had previously been demonstrated that p53 protein is aphysiologic target of the ATM kinase (Banin et al., supra, 1998; Canmanet al., supra, 1998), but most of the physiologic abnormalities in ATpatients and AT cells are not attributable to defects in signaling top53. Thus, it is clear that there must be other physiologic targets ofthis kinase. Building upon the assay described above (Example 2; Canmanet al., supra, 1998), we clarified the optimal conditions for in vitromeasurements of ATM kinase activity. These experiments demonstratedsignificant differences in the co-factor requirements of ATM and ATRcompared to the related kinase, DNA-PK. In particular, ATM and ATRrequire Mn²⁺, but not DNA ends or Ku proteins, for optimal in vitroactivity while DNA-PKcs requires Mg2+, DNA ends, and Ku proteins.Optimization of in vitro conditions to evaluate these three kinasesallows us to then use these in vitro assays for screening of potentialphysiologic targets of these enzymes.

[0258] Our general approach was to first identify peptide sequencesrecognized by these kinases in vitro, then extend these observations tofull-length proteins, followed by investigation of putative targets invivo. There are several advantages to this approach. First, it appearsthat the GST-peptides are much better than small synthetic peptides asin vitro substrates and it is easier and cheaper to make theoligonucleotides and clone them into the GST plasmid than to synthesizeevery peptide of interest. Second, easy alteration of the sequence ofthe oligonucleotides attached to the GST-linker allows us to quickly andeasily define a general consensus target motif and simultaneouslyidentify the putative phosphorylation site for each target. Theadvantage of immediately knowing the potential site of phosphorylationis exemplified by how quickly it allowed us to identify p95 as a truephysiologic substrate after the in vitro work had identified ser343 ofp95 as the likely target site and justified the effort required to makea site-specific, phosphoserine-specific antibody.

[0259] Third, comparing the abilities of the three related kinases tophosphorylate each of these substrates in vitro provides unexpectedclues about how they differ in in vivo function and providesdistinctions between the enzymes for development of specific inhibitorsof these kinases. As examples of insights that were not predictable apriori, our in vitro data suggests that Rad17 may be a physiologicsubstrate for ATR and not DNA-PK and that ligase IV may be a physiologicsubstrate for DNA-PK and not ATM or ATR. This latter possibility isparticularly intriguing because of the recently described role forligase IV in V(D)J recombination events that also involve DNA-PK (Franket al., Nature, 396:173, 1998). Building upon the preliminary consensustarget sequence for ATM generated from the p53ser15 mutagenesis work, wewere then able to identify some new potential substrates for ATM, ATRand DNA-PK including Rad17, Brca1, Brca2 p95, PTS, PhasI, WRN, DNA-5B,and Ligase IV. These targets represented the first screen of potentialsubstrates and additional proteins with reasonable ATM target sites arecontinuing to be evaluated.

[0260] It should be noted that the consensus target sequences wecharacterized should be considered as guidelines rather than concreterules. However, it does appear that glutamine at position N+1 appears isabsolutely required for activity of this kinase family and that nearbyhydrophobic amino acids, especially at positions N−3 and N−1, areimportant determinants. It is noted that since substitution of threoninefor serine had only a quantitative effect on phosphorylation by allthree enzymes, it is entirely conceivable that threonine could replaceserine as the targeted amino acid in certain protein targets.

[0261] ATM, p95 and Other Potential Substrates.

[0262] AT, with mutations in the ATM gene, and NBS, with mutations inthe p95/nibrin gene, share many phenotypic abnormalities, includingchromosomal instability, radiation sensitivity and defects in cell cyclecheckpoints in response to IR (Sbiloh, Ann. Rev. Genet., 31:635, 1997;Featherstone and Jackson, Curr. Biol., 8:R622, 1998). Thus, it wasreasonable to suspect that p95 and ATM might be involved in similarcellular processes in response to DNA damage responses and providedjustification for choosing this putative ATM target as the initialprotein suggested by our in vitro screen to characterize in vivo. ThisExample shows that ATM phosphorylates p95 on ser343 in vitro, that ATMcan bind to p95 in cells and that the phosphorylation of p95 in vivo inresponse to IR occurs on ser343 in an ATM-dependent manner. This set ofobservations is identical to those used to conclude that ATM is requiredfor phosphorylating ser15 of p53 in response to IR (Banin et al., supra,1998; Canman et al., supra, 1998).

[0263] At present, we do not know the functional significance of thisATM phosphorylation of p95Ser343 in response to IR. However, thisphysiologic linkage may in part explain how mutations in either the ATMor p95/nibrin genes result in similar, though not identical, phenotypes.p95 associates with hRad50 and hMre11, and this complex has beenimplicated in the recognition of DNA double strand breaks as well as therepair of DNA double strand breaks (Carney et al., Cell, 93:477, 1998;Varon et al., Cell, 93:467, 1998; Nelms et al., Science, 280:590, 1998;Trujillo et al., J. Biol. Chem., 273:21447, 1998; Paul and Gellert,Genes Dev., 13:1276, 1999). The corresponding protein complex inSaccharomyces cerevisiae, containing ScRad50, ScMre11, and ScXRS2, playsimportant roles in recombinational repair, meiotic recombination andtelomere maintenance (Fabre et al., Mol. Gen. Genet., 195:139, 1984;Ivanov et al., Genetics, 132:651, 1992; Johzuka and Ogawa, Genetics,139:1521, 1995; Tsukamoto et al., Genetics, 142:383, 1996; Moore andHaber, Mol. Cell Biol., 16:2164, 1996; Ohta et al., Proc. Natl. Acad.Sci USA, 95:646, 1998; Haber, Cell, 95:583, 1998; Usui et al., Cell,95:705, 1998; Chamankhah and Xiao, Nucl. Acids Res., 27:20729, 1999).Complexes of hMre11, Rad50, and p95 relocalize to DNA strand breakregions within 30 min after exposure of mammalian cells to IR,presumably to recognize the DNA damage (Nelms et al., supra, 1998), andform nuclear foci at 6 to 8 hours later, perhaps to facilitate DNArepair (Maser et al., Mol. Cell Biol., 17:6087, 1997; Carney et al.,supra, 1998). Interestingly, the formation of these foci is defective inNBS cells and has been reported to be measurably reduced in AT cells(Maser et al., supra, 1997).

[0264] These observations suggest that p95 is likely to be required forthe localization of the hMre11/hRad50 complex to DNA damage-inducedbreaks and that ATM dysfunction might alter the rate of the formation orstability of the hMre11/hRad50/p95 foci in response to IR. Ourobservations suggest that the link of ATM to this process may be throughphosphorylation of p95 on Ser343. The ATM target site in p95, Ser343, isimmediately adjacent to the BRCT domain in the N-terminal portion ofp95, which is believed to be involved in protein-protein interaction(Critchlow et al., Curr. Biol., 7:588, 1997; Saka et al., Genes Dev.,11:3387,1997; Yu et al., J. Biol. Chem., 273:25388, 1998; Li et al.,Oncogene, 18:1689, 1999; Soulier and Lowndes, Curr. Biol., 9:551, 1999).Thus, it is also possible that phosphorylation of p95 on ser343 by ATMaffects its interaction with other proteins.

[0265] A direct role for ATM in repair of DNA breaks has been difficultto clarify. Measurements of religation of DNA breaks in AT cells afterIR is not reproducibly abnormal (McKinnon, Hum. Genet., 75:197, 1987)nor is there an obvious DSB repair deficiency in NBS cells (Nove et al.,Mutat. Res., 163:225, 1986; Kraakman-van der Zwet et al., Mutat. Res.,434:17, 1999). However, many of the phenotypic abnormalities in A-Tcells and NBS cells clearly indicate a problem with response to DNAbreaks. These abnormalities include a high frequency of chromosomaltranslocations or inversions involving chromosomes 7 and 14 in T-cells(which contain sites of V(D)J recombination), increased telomere fusionsand accelerated telomere shortening, high numbers of chromosomal gapsand breaks after IR, defects in meiotic recombination, and radiationsensitivity (Lavin and Shiloh, Ann. Rev. Immunol., 15:177, 1997; Shiloh,Ann. Rev. Genet., 31:635, 1997). The observation that ATM phosphorylatesp95 in response to ionizing radiation now demonstrates that theseproteins function in the same DNA damage response pathway.

[0266] Other potential targets that we identified in our screens coulduncover other roles for ATM in DNA repair processes. Patients withheterozygous germline mutations in the BRCA1 gene have a markedlyincreased risk of developing breast cancer (Futreal et al., Science,266:120, 1994; Miki et al., Science, 266:66, 1994) and it has beensuggested that heterozygous germline ATM mutations also increase breastcancer risk (Swift et al., N. Engl. J. Med., 316:1289, 1987; Swift etal., N. Engl. J. Med., 325:1831, 1991; Lavin, BMJ, 371:486, 1998; Swiftand Su, BMJ, 318:400, 1999). Additionally, it appears that Brca1participates both in transcription as a transcription factor andDNA-repair through association with Rad51/Brca2 (Scully et al., Cell,88:265, 1997; Somasundaram et al., Nature, 389:187, 1997; Chen et al.,Mol. Cell, 2:317, 1998). Using an approach similar to that used for p95,further studies can investigate the degree to which Brca1 is an in vivotarget of ATM and the physiologic significance of such aphosphorylation.

[0267] The function of the mammalian Rad17 protein is currently unknown,but data from yeast implicate this protein in DNA damage checkpointcontrol (Bao et al., Cell Growth Differ., 9:961, 1998; Bluyssen et al.,Genomics, 55:219, 1999; Li et al., supra, 1999). Elucidation of theextent to which this is a physiologic target of either the ATM or ATRkinases may shed additional light on regulation of mammalian DNA repairprocesses.

[0268] Finally, since ATM kinase activity is enhanced by DNA breakage(Banin et al., supra, 1998; Canman et al., supra, 1998), it appears thatthe potential ATM autophosphorylation site (ATM440) identified hereincontributes to regulation of its activity. The studies provide initialinsights into understanding AT and signaling pathways involving ATM andprovide a paradigm for further studies of this kinase family andpotentially other kinases.

Example 4 Identification of Brca1 as an in vitro ATM Target

[0269] Taking similar approaches to that of Example 3 with a number ofother proteins has yielded preliminary data showing that the breastcancer susceptibility gene product, Brca1, is also a physiologic targetof the ATM kinase. All of the potential ATM target sites (based on ourconsensus sequence) in Brca1 were tested and the data suggest that thereare two sites in Brca1 phosphorylated by ATM. The results have potentialsignificance for both cancer causation and new cancer therapeuticapproaches.

Example 5 Role of ATM in Insulin Signaling

[0270] We have further investigated the role of ATM in insulinsignaling. As discussed above, ATM appears to be critical for insulinsignaling in adipocytes. Experimental evidence supports this. First,PhasI/4E-BP1 (but not 4E-BP2) is a good substrate for ATM, and itsphosphorylation site has been mapped. As shown in Example 3, serine 94of human PhasI/4E-BP1 (equivalent to serine 93 of rat PhasI/4E-BP1) is agood in vitro peptide substrate for ATM. However, unexpectedly, ATM isable to phosphorylate full-length 4E-BP1 mutated at serine 94, thussuggesting that another amino acid is a relevant target in thefull-length protein. Using the ATM target consensus, we identifiedanother potential ATM phosphorylation site in 4E-BP1 and then showedthat ATM actually phosphorylates serine 111 of the protein.

[0271] In vitro kinase assays were used to show that ATM is capable ofphosphorylating GST-petides containing either an 18 amino acid region ofthe protein around serine 93 or around serine 111 of 4E-BP1. ATM canphosphorylate full-length 4E-BP1 as well as 4E-BP1 mutated at serine 93,but cannot phosphorylate 4E-BP 1 mutated at serine 111. Thisdemonstrates that serine 111 is the site of phosphorylation by ATM in4E-BP1.

[0272] A recent manuscript identified serine 111 of 4E-BP1 as animportant insulin-induced in vivo phosphorylation site in adipocytes(Hessom, et al., Journal of Biochemistry, 336:39-48, 1998), which lendsfurther importance to this observation. We have since extended theseobservations to demonstrate that insulin treatment of adipocytesdirectly activates the ATM kinase (FIG. 3).

[0273] ATM was immunoprecipitated from 3T3-L1 after differentiation andwithout (−) or with (+) exposure to insulin. The immunoprecipitated ATMwas then used in an in vitro kinase assay with GST-4E-BP1 protein as thesubstrate. Equivalent amounts of ATM and GST-4E-BP1 were used. Insulintreatment significantly enhanced ATM kinase activity in thesedifferentiated cells, when normalized to standards for the amount of4E-BP1 protein.

[0274] Further data demonstrated the physiologic importance of thisprocess by showing that ATM activity is necessary for insulin inducedrelease of 4E-BP 1 from eIF-4E. Fibroblasts from normal or AT mice weretreated with (+) or without (−) insulin and total cellular and complexedeIF-4E and 4E-BP1 were examined by Western blot. Total cellular eIF-4Eand 4E-BP1 were unchanged by insulin treatment. The amount of 4E-BP1bound to eIF-4E was examined by immunoprecipitating eIF-4E with amethyl-G column and then blotting for both proteins. Insulin caused arelease of 4E-BP1 from eIF-4E in normal cells (A29), but much less4E-BP1 was released in AT cells (A38). This result was repeated in humanfibroblasts expressing a dominant-negative form of ATM.

[0275] Finally, we also showed that mutation of the ATM target site in4E-BP1, serine 111, also abrogates insulin-mediated release of 4E-BP1from eIF-4E. This release of 4E-BP1 is considered necessary for insulinstimulation of adipocyte growth.

[0276] Thus, these observations provide further significant evidencesupporting the role of ATM in insulin signaling in adipocytes and thepotential utility of ATM inhibition in the treatment of obesity.Furthermore, it illustrates yet another example of how the in vitroscreening assay and clarification of a consensus target sequence for ATMof this invention provide critical biological insights.

Example 6 IGF-I Activates ATM

[0277] Since the growth factor and survival factor insulin-like growthfactor (IGF)-I activates cellular signaling in a manner similar toinsulin, the insulin observations above led us to also investigatewhether IGF-I might activate ATM kinase. Since IGF-I is a neuronalsurvival factor and since patients with mutated ATM exhibit neuronaldegeneration, we were particularly intrigued by the possibility that ATMactivation by IGF-I might be cell-type specific. Previous resultsdemonstrated that the neuroblastoma cell line, SY5Y, responds to IGF-I,and this cell line can be differentiated along a neuronal lineage in invitro culture with retinoic acid. Undifferentiated and differentiatedSY5Y cells were treated with IGF-I and ATM activation was assessed.

[0278] ATM was immunoprecipitated from either undifferentiated ordifferentiated SY5Y neuroblastoma cells after treatment with 10 nM IGF-I(control cells were untreated). The immunoprecipitated ATM was then usedin an in vitro kinase assay with the GST-p53 protein as the substrate.Equivalent amounts of ATM and GST-p53 were used. IGF-I treatmentsignificantly enhanced ATM kinase activity in the differentiated, butnot undifferentiated, cells.

[0279] This observation, derived from the technologies described above,provides new insights into a novel mechanism of neurodegeneration. Wecan now identify the physiologic substrates of the ATM kinase in thissetting using the in vitro assays and consensus sequence and then followup in vitro leads with in vivo studies, as with the p95/nibrin targetdescribed above (Example 3).

Example 7 Use of the ATM Kinase Assay For Identification of an ATMInhibitor

[0280] A useful application of the in vitro assay for ATM kinaseactivity disclosed herein is for screening compounds which inhibit ATMkinase activity. Such compounds would then be candidate compounds forradiosensitization of tumors in vivo. The in vitro assay described above(Example 1) utilizes the recombinant ATM kinase protein (usually with anepitope-tag for ease of purification) and an identified substrate, suchas the sequence in the amino-terminal domain of p53 protein. Adding acompound that is a candidate inhibitor blocks or significantly reduces(compared to the control level of phosphorylation without the candidateinhibitor) the ability of the recombinant ATM protein to phosphorylatethis substrate in this assay. The readout for the level of ATM-mediatedphosphorylation may be incorporation of radioactive phosphate, thoughother options for readouts of kinase activity are also available.Development of this type of in vitro assay is particularly convenientfor drug screens because it can be set up in automated high throughputscreening assays. For example, the substrate can be attached to wells inmicrotiter dishes. Recombinant ATM enzyme can be added to the wells withappropriate buffers and co-factors for kinase reaction, and candidateinhibitory compounds can be added to selected wells to evaluate oneswhich inhibit the kinase activity. As a further control, kinase-dead ATMprotein can be added to certain wells to further demonstrate specificityin these assays. Use of the identified optimal peptide consensus sitesas targets for ATM activity can further enhance the sensitivity andspecificity of the assay. In addition, if small peptides by themselves(rather than as GST-conjugated peptides) are successfully used in thisassay, then the ability to utilize this drug screening approach on alarge scale is further enhanced because the peptide substrate can bemade synthetically rather than by recombinant methods.

[0281] The present invention is not to be limited in scope by thespecific embodiments described herein. Indeed, various modifications ofthe invention in addition to those described herein will become apparentto those skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

[0282] It is further to be understood that all base sizes or amino acidsizes, and all molecular weight or molecular mass values areapproximate, and are provided for description.

[0283] All patents, patent applications, publications, and othermaterials cited herein are hereby incorporated herein reference in theirentireties.

1 49 1 7 PRT Artificial Sequence PHOSPHORYLATION 4 Xaa at position oneis a hydrophobic amino acid. Xaa at position two is any amino acid. Xaaat position three is a hydrophobic amino acid or Asp. Xaa at positionsix is any amino acid. 1 Xaa Xaa Xaa Ser Gln Xaa Xaa 1 5 2 7 PRTArtificial Sequence PHOSPHORYLATION 4 Xaa at position seven is any aminoacid. 2 Pro Pro Asp Ser Gln Glu Xaa 1 5 3 7 PRT Artificial SequencePHOSPHORYLATION 4 Xaa at position seven is any amino acid. 3 Leu Pro LeuSer Gln Asp Xaa 1 5 4 7 PRT Artificial Sequence PHOSPHORYLATION 4 Xaa atposition seven is any amino acid. 4 Leu Pro Leu Ser Gln Pro Xaa 1 5 5 7PRT Artificial Sequence PHOSPHORYLATION 4 Xaa at position seven is anyamino acid. 5 Leu Pro Ala Ser Gln Asp Xaa 1 5 6 7 PRT ArtificialSequence PHOSPHORYLATION 4 Xaa at position seven is any amino acid. 6Leu Pro Ala Ser Gln Pro Xaa 1 5 7 14 PRT Homo sapiens 7 Ser Val Glu ProPro Leu Ser Gln Glu Thr Phe Ser Asp Leu 1 5 10 8 14 PRT Homo sapiens 8Val Leu Ser Pro Leu Pro Ser Gln Ala Met Asp Asp Leu Met 1 5 10 9 14 PRTHomo sapiens 9 Glu Pro Pro Met Glu Ala Ser Gln Ser His Leu Arg Asn Ser 15 10 10 14 PRT Homo sapiens 10 Thr Pro Gly Pro Ser Leu Ser Gln Gly ValSer Val Asp Glu 1 5 10 11 14 PRT Homo sapiens 11 Gln Gln Leu Phe Tyr IleSer Gln Pro Gly Ser Ser Val Val 1 5 10 12 14 PRT Homo sapiens 12 Phe SerVal Leu Arg Phe Ser Gln Lys Phe Val Asp Arg Val 1 5 10 13 14 PRT Homosapiens 13 Arg Ala Arg Ala Leu Arg Ser Gln Ser Glu Glu Ser Ala Ser 1 510 14 14 PRT Homo sapiens 14 Ser Ala Ser Arg Gly Gly Ser Gln Arg Gly ArgAla Phe Lys 1 5 10 15 14 PRT Homo sapiens 15 Ser Ser Ser Lys Ile Met SerGln Ser Gln Val Ser Lys Gly 1 5 10 16 14 PRT Homo sapiens 16 Asn Val LysTyr Ser Ser Ser Gln Pro Glu Pro Arg Thr Gly 1 5 10 17 14 PRT Homosapiens 17 Val Gln Gly Ile Ser Phe Ser Gln Pro Thr Cys Pro Asp His 1 510 18 14 PRT Homo sapiens 18 Thr Trp Ser Leu Pro Leu Ser Gln Asp Ser AlaSer Glu Leu 1 5 10 19 14 PRT Homo sapiens 19 Ala Ser Glu Leu Pro Ala SerGln Pro Gln Pro Phe Ser Ala 1 5 10 20 14 PRT Homo sapiens 20 Thr Ile GlyMet His Leu Ser Gln Ala Val Lys Ala Gly Cys 1 5 10 21 14 PRT Homosapiens 21 Glu Lys Ala Tyr Ser Ser Ser Gln Pro Val Ile Ser Ala Gln 1 510 22 14 PRT Homo sapiens 22 Thr Val Glu Pro Ile Ile Ser Gln Leu Val ThrVal Leu Leu 1 5 10 23 14 PRT Homo sapiens 23 Tyr Pro Gly Ile Asp Leu SerGln Val Tyr Glu Leu Leu Glu 1 5 10 24 14 PRT Homo sapiens 24 Pro Leu LeuMet Ile Leu Ser Gln Leu Leu Pro Gln Gln Arg 1 5 10 25 14 PRT Homosapiens 25 Tyr Lys Val Val Pro Leu Ser Gln Arg Ser Gly Val Leu Glu 1 510 26 14 PRT Homo sapiens 26 Asp Leu Leu Met Tyr Leu Ser Gln Leu Val GlnAla Leu Lys 1 5 10 27 14 PRT Homo sapiens 27 Arg Leu Arg Pro Leu Leu SerGln Leu Gly Gly Asn Ser Val 1 5 10 28 14 PRT Homo sapiens 28 Cys Arg AlaPro Glu Val Ser Gln His Val Tyr Gln Ala Tyr 1 5 10 29 14 PRT Homosapiens 29 Lys Val Ser Pro Tyr Leu Ser Gln Phe Gln Gln Asp Lys Gln 1 510 30 14 PRT Homo sapiens 30 Leu Pro Cys Leu Glu Ile Ser Gln Ser Val ThrGly Phe Gly 1 5 10 31 14 PRT Homo sapiens 31 Ala Pro Glu Pro Pro Val SerGln Glu Leu Pro Cys Ser Arg 1 5 10 32 14 PRT Homo sapiens 32 Ser Ala SerLeu Phe Ser Ser Gln Cys Ser Glu Leu Glu Asp 1 5 10 33 14 PRT Homosapiens 33 Asp Cys Ser Gly Leu Ser Ser Gln Ser Asp Ile Leu Thr Thr 1 510 34 14 PRT Homo sapiens 34 Ser Ser Glu Tyr Pro Ile Ser Gln Asn Pro GluGly Leu Ser 1 5 10 35 14 PRT Homo sapiens 35 Leu Leu His Ile Pro Val SerGln Phe Lys Asp Ala Asp Leu 1 5 10 36 14 PRT Homo sapiens 36 Gly Ala SerPro Val Leu Ser Gln Gly Val Asp Pro Arg Ser 1 5 10 37 14 PRT Homosapiens 37 Trp Glu Thr Pro Asp Leu Ser Gln Ala Glu Ile Glu Gln Lys 1 510 38 14 PRT Homo sapiens 38 Gln Pro Glu Pro Pro Val Ser Gln Ser Glu IleSer Glu Pro 1 5 10 39 14 PRT Homo sapiens 39 Asp Leu Lys Leu Gly Val SerGln Gln Thr Ile Phe Ser Val 1 5 10 40 14 PRT Homo sapiens 40 Phe Pro AlaPro Ala Pro Ser Gln Ala Glu Lys Lys Ser Arg 1 5 10 41 14 PRT Homosapiens 41 Ile Val Pro Cys Thr Ile Ser Gln Leu Leu Ser Ala Thr Leu 1 510 42 14 PRT Homo sapiens 42 Thr Gly Asn Val Glu Ile Ser Gln Val Thr IleVal Gly Ile 1 5 10 43 9 PRT Homo sapiens 43 Glu Leu Lys Pro Val Leu SerGln Gly 1 5 44 14 PRT Homo sapiens 44 Asp Pro Gly Ala Asp Leu Ser GlnTyr Lys Met Asp Val Thr 1 5 10 45 14 PRT Homo sapiens 45 Ser Pro Leu AlaPro Val Ser Gln Gln Gly Trp Arg Ser Ile 1 5 10 46 36 DNA ArtificialSequence PCR primer 46 tccccaggaa ttcccggcca tcccagtaca ggatta 36 47 36DNA Artificial Sequence PCR primer 47 tgcggccgct cgagtttttt gttccattttggagac 36 48 45 DNA Artificial Sequence PCR primer 48 gaatccctcgagcctaccgc catgtggaaa ctgctgcccg ccgcg 45 49 48 DNA Artificial SequencePCR primer 49 gtcgacgagc ggccgccacc tcagggatct tctccttttt aaataagg 48

What is claimed is:
 1. A method for identifying an ATM kinase substraterecognition sequence in a protein, which comprises contacting an ATMkinase with a fusion polypeptide and detecting whether binding hasoccurred between the ATM kinase and the fusion polypeptide, wherein thefusion polypeptide contains a structural portion and a candidate ATMkinase substrate recognition sequence portion.
 2. The method accordingto claim 1, wherein the structural portion lacks an ATM kinaserecognition sequence.
 3. The method according to claim 1, wherein thecandidate sequence has an ATM kinase recognition sequence motif,including a serine, whereby the polypeptide is phosphorylatable by theATM kinase on the serine.
 4. The method according to claim 1, whereinthe candidate sequence has a modified ATM kinase recognition sequencemotif lacking an amino acid residue phosphorylatable by ATM kinase,whereby the polypeptide is not phosphorylatable by the ATM kinase. 5.The method according to claim 1, wherein the structural portion is GST.6. The method according to claim 1, wherein the candidate peptidesequence is selected from the group consisting of: SVEPPLSQETFSDL (SEQID NO:7); TPGPSLSQGVSVDE (SEQ ID NO:10); QQLFYISQPGSSVV (SEQ ID NO:11);EPPMEASQSHLRNS (SEQ ID NO:9); NVKYSSSQPEPRTG (SEQ ID NO:16);KAYSSSQPVISAQ (SEQ ID NO:21); VQGISFSQPTCPDH (SEQ ID NO:17);WETPDLSQAEIEQ (SEQ ID NO:37); GASPVLSQGVDPR (SEQ ID NO:36);PLLMILSQLLPQQR (SEQ ID NO:24); DCSGLSSQSDILTT (SEQ ID NO:33);TWSLPLSQDSASEL (SEQ ID NO:18); and ASELPASQPQPFSA (SEQ ID NO:19).


7. A method for identifying a putative ATM target protein, which methodcomprises analyzing the sequence of the protein to determine whether itcontains a sequence corresponding to an ATM substrate recognitionconsensus sequence motif B₁-X-B₂-S-Q-X-X (SEQ ID NO:1), wherein B₁ is ahydrophobic amino acid, B₂ is a hydrophobic amino acid or aspartic acid,X is any amino acid, Q is glutamine, and S is serine.
 8. The methodaccording to claim 7, wherein the ATM substrate recognition sequencemotif is selected from the group consisting of P-P-D-S-Q-E-X (SEQ IDNO:2) and L-P-[L or A]-S-Q-[D or P]-X (SEQ ID NO:3), wherein P isproline, D is aspartic acid, E is glutamic acid, L is leucine, and A isalanine.
 9. The method according to claim 7, wherein the target proteinis involved in a cellular process selected from the group consisting ofdouble stranded DNA break repair, telomere synthesis or repair, theaging process, tumor suppression, insulin signaling, insulin-like growthfactor-I signaling, cell cycle control, affecting cell survival afterHTLV infection and autophosphorylation.
 10. The method according toclaim 7, further comprising determining whether the sequencecorresponding to an ATM substrate recognition consensus sequence motifis phosphorylated by ATM.
 11. The method according to claim 10, whereindetermining whether the sequence corresponding to an ATM substraterecognition consensus sequence motif is phosphorylated by ATM comprisescontacting ATM kinase with a fusion polypeptide and detecting whetherthe fusion polypeptide is phosphorylated, wherein the fusion polypeptidecontains a structural portion and the sequence corresponding to an ATMsubstrate recognition consensus sequence motif.
 12. The method accordingto claim 10, wherein determining whether the sequence corresponding toan ATM substrate recognition consensus sequence motif is phosphorylatedby ATM comprises contacting ATM kinase with the target protein anddetecting whether the target protein is phosphorylated.
 13. A method foridentifying an ATM-regulated process comprising: a) identifying a targetprotein comprising an ATM substrate recognition phosphorylation sequenceaccording to the method of claim 7; b) modulating ATM-mediatedphosphorylation of the target protein; and c) determining whethermodulation of ATM-mediated phosphorylation of the target protein affectsa pathway, which indicates that the process is an ATM-regulated process.14. The method according to claim 13, wherein the ATM-regulated pathwayis selected from the group consisting of double stranded DNA breakrepair, telomere synthesis or repair, the aging process, tumorsuppression, insulin signaling, insulin-like growth factor-I signaling,cell cycle control, affecting cell survival after HTLV infection andautophosphorylation.
 15. The method according to claim 13, wherein thetarget protein is selected from the group consisting of NBS/p95, MRE11,PHASI, CHK1, Werner, PST1, CUT1, ATM, BRCA1, and RAD17.
 16. The methodaccording to claim 13, wherein modulation of ATM-mediatedphosphorylation comprises inhibiting ATM-mediated phosphorylation. 17.The method according to claim 13, wherein modulation of ATM-mediatedphosphorylation comprises increasing ATM-mediated phosphorylation.
 18. Amethod for modulating an ATM-regulated process comprising modulatingATM-mediated phosphorylation of a target protein comprising an ATMkinase substrate recognition sequence in a cell.
 19. The methodaccording to claim 18, wherein the modulation of ATM-mediatedphosphorylation comprises inhibiting ATM-mediated phosphorylation. 20.The method according to claim 19, wherein inhibiting ATM-mediatedphosphorylation comprises expressing kinase dead ATM mutant in the cell.21. The method according to claim 18, wherein modulation of ATM-mediatedphosphorylation comprises increasing ATM-mediated phosphorylation. 22.The method according to claim 21, wherein the ATM-mediatedphosphorylation is increased by increasing the level of expression ofATM in a cell.
 23. The method according to claim 18, wherein theATM-regulated process is selected from the group consisting of doublestranded DNA break repair, telomere synthesis or repair, the agingprocess, tumor suppression, insulin signaling, insulin-like growthfactor-I signaling, cell cycle control, affecting cell survival afterHTLV infection and autophosphorylation.
 24. The method according toclaim 18, wherein the target protein is selected from the groupconsisting of NBS/p95, MRE11, PHASI, CHK1, Werner, PST1, CUT1, ATM,BRCA1, and RAD17.
 25. A nucleic acid encoding a kinase dead ATM mutant.26. A recombinant vector which codes for expression of a defective ATMpolypeptide.
 27. The recombinant vector of claim 26 wherein thedefective ATM polypeptide is a kinase dead ATM mutant.
 28. A recombinantcell line containing the vector of claim
 26. 29. A recombinant vectorwhich codes on expression for a fusion polypeptide, wherein the fusionpolypeptide contains a structural portion and a candidate ATM kinaserecognition sequence portion.
 30. The vector according to claim 29,wherein the structural portion lacks an ATM kinase recognition sequence.31. The vector according to claim 29, wherein the fusion peptide is aGST fusion peptide.
 32. The vector according to claim 29, wherein thecandidate peptide comprises a sequence selected from the groupconsisting of: SVEPPLSQETFSDL (SEQ ID NO:7); TPGPSLSQGVSVDE (SEQ IDNO:10); QQLFYISQPGSSVV (SEQ ID NO:11); EPPMEASQSHLRNS (SEQ ID NO:9);NVKYSSSQPEPRTG (SEQ ID NO:16); EKAYSSSQPVISAQ (SEQ ID NO:21);VQGISFSQPTCPDH (SEQ ID NO:17); WETPDLSQAEIEQ (SEQ ID NO:37);GASPVLSQGVDPR (SEQ ID NO:36); PLLMILSQLLPQQR (SEQ ID NO:24);DCSGLSSQSDILTT (SEQ ID NO:33); TWSLPLSQDSASEL (SEQ ID NO:18); andASELPASQPQPFSA (SEQ ID NO:19).


33. A method for screening for a compound that modulates ATM-mediatedphosphorylation, comprising detecting whether there is a change in thelevel of ATM-mediated phosphorylation of a polypeptide comprising an ATMsubstrate recognition sequence in the presence of a candidate compound,wherein an increase in the level of phosphorylation indicates that thecompound agonizes ATM-mediated phosphorylation, and a decrease in thelevel of phosphorylation indicates that the compound antagonizesATM-mediated phosphorylation.
 34. The method according to claim 33,wherein the compound selectively modulates ATM-mediated phosphorylation.35. The method according to claim 33, further comprising detectinginhibition of a cellular process mediated by ATM phosphorylation of atarget protein, wherein inhibition of the activity is indicative ofinhibition of ATM.
 36. The method according to claim 35, wherein thechange of a cellular process is selected from the group consisting ofloss of S-phase checkpoint, a defect in the G₂/M checkpoint, an increasein radiosensitivity, and increase in sensitivity to chemotherapeuticagents.
 37. A method for screening for a compound that induces anATM-regulated pathway in a cell, comprising contacting the cell with acandidate compound, and detecting whether the ATM-mediated process isinduced in the cell, wherein the cell is defective for expression ofATM, with the proviso that the pathway is not tumor suppression or cellcycle control.
 38. The method according to claim 33, wherein screeningfor a compound that induces an ATM-regulated process in a cell linecomprising contacting a cell line that empresses an ATM kinase deadmutant with a candidate compound, and detecting whether the ATM-mediatedpathway is induced in the cell line.
 39. A composition comprising ATMand a polypeptide, wherein the polypeptide comprises an ATM kinasesubstrate recognition sequence.
 40. The composition of claim 39, whereinthe ATM kinase substrate recognition sequence is selected from the groupconsisting of SVEPPLSQETFSDL (SEQ ID NO:7); TPGPSLSQGVSVDE (SEQ IDNO:10); QQLFYISQPGSSVV (SEQ ID NO:11); EPPMEASQSHLRNS (SEQ ID NO:9);NVKYSSSQPEPRTG (SEQ ID NO:16); EKAYSSSQPVISAQ (SEQ ID NO:21);VQGISFSQPTCPDH (SEQ ID NO:17); WETPDLSQAEIEQ (SEQ ID NO:37);GASPVLSQGVDPR (SEQ ID NO:36); PLLMILSQLLPQQR (SEQ ID NO:24);DCSGLSSQSDILTT (SEQ ID NO:33); TWSLPLSQDSASEL (SEQ ID NO:18); andASELPASQPQPFSA (SEQ ID NO:19).


41. A peptide comprising a sequence corresponding to an ATM substraterecognition consensus sequence motif B₁-X-B₂-S-Q-X-X (SEQ ID NO: 1),wherein B₁ is a hydrophobic amino acid, B₂ is a hydrophobic amino acidor aspartic acid, X is any amino acid, Q is glutamine, and S is serine.42. The peptide of claim 41, wherein the ATM substrate recognitionsequence motif is selected from the group consisting of P-P-D-S-Q-E-X(SEQ ID NO:2) and L-P-[L or A]-S-Q-[D or P]-X (SEQ ID NO:3), wherein Pis proline, D is aspartic acid, E is glutamic acid, L is leucine, and Ais alanine.
 43. The peptide of claim 41, wherein the ATM kinasesubstrate recognition sequence is selected from the group consisting ofSVEPPLSQETFSDL (SEQ ID NO:7); TPGPSLSQGVSVDE (SEQ ID NO:10);QQLFYISQPGSSVV (SEQ ID NO:11); EPPMEASQSHLRNS (SEQ ID NO:9);NVKYSSSQPEPRTG (SEQ ID NO:16); EKAYSSSQPVISAQ (SEQ ID NO:21);VQGISFSQPTCPDH (SEQ ID NO:17); WETPDLSQAEIEQ (SEQ ID NO:37);GASPVLSQGVDPR (SEQ ID NO:36); PLLMILSQLLPQQR (SEQ ID NO:24);DCSGLSSQSDILTT (SEQ ID NO:33); TWSLPLSQDSASEL (SEQ ID NO:18); andASELPASQPQPFSA (SEQ ID NO:19).


44. A method for identifying a modulator of ATM-mediated activitycomprising determining the extent of HTLV integration in the presence ofa potential modulator of ATM-mediated activity, wherein the absence ofHTLV integration indicates that said potential modulator is a modulatorof ATM-mediated activity.
 45. A method for inhibiting HTLV integrationcomprising contacting a cell with an ATM inhibitor.
 46. The methodaccording to claim 18, wherein the ATM-regulated process is affectingcell survival after HTLV infection.
 47. The method according to claim18, wherein the ATM-regulated process is insulin-like growth factor-Isignaling.